Pd-1 single domain antibodies and therapeutic compositions thereof

ABSTRACT

Provided herein are binding polypeptides that specifically bind PD-1. More specifically, provided herein are fusion proteins, including multivalent and/or multispecific constructs and chimeric antigen receptors, that bind PD-1. Also provided are pharmaceutical compositions containing the polypeptides, nucleic acid molecules encoding the polypeptides and vectors and cells thereof, and methods of use and uses of the provided PD-1 binding polypeptides for treating diseases and conditions, such as cancer.

RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 16/600,298,filed Oct. 11, 2019, which claims the benefit of U.S. ProvisionalApplication No. 62/744,615 filed on Oct. 11, 2018, and U.S. ProvisionalApplication No. 62/791,152 filed on Jan. 11, 2019, the contents of whichare incorporated herein by reference in their entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filenameINHI-041-D01US_SeqList_ST25, dated recorded: Nov. 8, 2021; 305kilobytes).

FIELD

This disclosure generally provides binding polypeptides thatspecifically bind PD-1. More specifically, the disclosure relates tofusion protein, including multivalent and/or multispecific constructsand chimeric molecules, that bind at least PD-1. The disclosure alsoprovides nucleic acid molecules encoding the polypeptides and vectorsand cells thereof, and methods of use and uses of the provided PD-1binding polypeptides for treating diseases and conditions, such ascancer.

BACKGROUND

PD-1 is a member of the immunoglobulin superfamily of immune cellmodulating molecules. It is expressed on the surface of a activatedT-cells. The expression of PD-1 on activated T-cells is targeted byPD-L1 by tumor cells and stromal cells in the tumor microenvironment tosuppress immune responses, thereby making agents that block or targetPD-1 a desirable therapeutic target. Improved therapeutic molecules andagents targeting PD-1 are needed. Provided herein are embodiments thatmeet such needs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G depict the binding of 18H10 and its humanized varianthz18H10, or 1-14 to FreeStyle 293 cells expressing human PD-1 (FIG. 1A,FIG. 1D, FIG. 1E, FIG. 1F, and FIG. 1G), cynomolgus PD-1 (FIG. 1B) ormouse PD-1 (FIG. 1C). Binding to untransfected (293) cells also wasassessed and is shown.

FIG. 2 is a graph depicting the binding of 18H10 and its humanizedvariant hz18H10v7 to activated human T cells. Test article binding toactivated T cells was quantified by flow cytometry.

FIGS. 3A-3B is a graph depicting the ability of 18H10, its humanizedvariant hz18H10v7 or 1-14 to block PD1/PDL1-mediated suppression of Tcell receptor (TCR) signaling in a Jurkat reporter luciferase assaysystem. FIG. 3A depicts the PD1 blockade by 18H10 and hz18H10v7, whileFIG. 3B depicts the PD1 blockade by 1-14.

DETAILED DESCRIPTION

Provided herein are polypeptides that specifically bind to PD-1,hereinafter also called PD-1-binding polypeptides. In some embodiments,the provided binding polypeptides comprise at least one VHH domain thatbinds PD-1. In some embodiments, a PD-1-binding polypeptide providedherein comprises one, two, three, four, five, six, seven, or eight VHHdomains that each individually bind PD-1. In some embodiments, aPD-1-binding polypeptide provided herein comprises one, two, three, orfour VHH domains that bind PD-1. In some embodiments, the PD-1-bindingpolypeptides are monospecific. In some embodiments, the PD-1-bindingpolypeptides are multispecific. For example, provided PD-1-bindingpolypeptides include polypeptides that may comprise at least one VHHdomain that binds PD-1 and one or more additional binding domains, suchas one or more additional VHH domains, that bind one or more targetproteins other than PD-1.

In some embodiments, a PD-1-binding polypeptide comprises at least oneVHH domain that binds PD-1 and an Fc domain. In some embodiments, aPD-1-binding polypeptide provided herein comprises one, two, three, orfour VHH domains that bind PD-1 and an Fc domain. In some embodiments,an Fc domain mediates dimerization of the PD-1-binding polypeptide atphysiological conditions such that a dimer is formed that doubles thenumber of PD-1 binding sites. For example, a PD-1-binding polypeptidecomprising three VHH domains that bind PD-1 and an Fc region istrivalent as a monomer, but at physiological conditions, the Fc regionmay mediate dimerization, such that the PD-1-binding polypeptide existsas a hexavalent dimer under such conditions.

Programmed cell death protein-1 (PD-1) is a type I membrane protein andis a member of the extended CD28/CTLA-4 family of T cell regulators PD-1(The EMBO Journal (1992), vol. 11, issue 11, p. 3887-3895). It isreported that PD-1 expression in periphery is observed in myeloid cellsincluding T cells or B lymphocytes activated by stimulation from antigenreceptors, or activated macrophages (International Immunology (1996),vol. 18, issue 5, p. 765-772). Expression of PD-1 on the cell surfacehas also been shown to be upregulated through IFN-γ stimulation. Twocell surface glycoprotein ligands for PD-1 have been identified, PD-1and PDL-2, and have been shown to down-regulate T cell activation andcytokine secretion upon binding to PD-1 (Freeman et al. (2000) J. Exp.Med. 192:1027-34; Latchman et al. (2001) Nat. Immunol. 2:261-8; Carteret al. (2002) Eur. J. Immunol. 32:634-43; Ohigashi et al. (2005) Clin.Cancer Res. 11:2947-53). Both PD-1 (B7-H1) and PD-L2 (B7-DC) are B7homologs that bind to PD-1. PD-L1 and PD-L2 are normally expressed onthe surface of T cells, B cells, and myeloid cells. PD-L1 and PD-L2 arenegative regulators of immune activation and are capable ofdown-modulating the immune response via interactions with PD-1 receptor.In some aspects, PD-1 is expressed on NK cells and T cells, includingCD4+ and CD8+ T cells, whereby engagement of PD-1 can inhibit activationcell activation, proliferation, and/or expansion.

An exemplary sequence of human PD-1 is set forth as follows:

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO: 286, signal sequence underlined)

In some cases, the provided PD-1 binding polypeptides directly block orinhibit the interaction between PD-L1/L2 and PD-1. In some embodiments,provided molecules that inhibit or reduce the interaction between PD-L1and/or PD-L2 and PD-1 modulate immune responses. While transmission ofan inhibitory signal may lead to downmodulation in immune cell responses(and a resulting downmodulation in the overall immune response),blocking an inhibitory signal in immune cells leads to upmodulation ofimmune cell responses (and a resulting upmodulation of an immuneresponse). In some cases, modulation by enhancement of an immuneresponse can be used to treat certain disease or conditions in which theimmune response is suppressed, such as cancers. In some embodiments, theprovided PD-1 binding polypeptides can be used as a therapeutic toinhibit or reduce tumor cell growth or survival.

A variety of PD-1 polypeptide binding formats are provided. In someembodiments, a PD-1 polypeptide provided herein is bivalent, such as byfusion with an Fc protein. In some examples, PD-1 binding polypeptidesinclude PD-1 VHH-Fc polypeptides. In some embodiments, the Fc is an Fcthat exhibits immune effector activity, such as one or more effectorfunctions such as antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP) and/orcomplement-dependent cytotoxicity (CDC). In other embodiments, the PD-1polypeptide may be a multispecific polypeptide containing at least oneadditional molecule. In some embodiments, the additional molecule iscapable of engaging with another molecule in a tumor-associatedmicroenvironment, such as with a tumor associated antigen or an immunecell, e.g. a T cell. In particular embodiments, the provided PD-1polypeptide binding formats block the interaction between PD-1 and PD-L1and/or PD-L1 and/or reduce, inhibit or suppress the inhibitory signalmediated by PD-1 in a cell, such as a T cell.

In some embodiments, the provided PD-1-binding polypeptides can be usedto stimulate an immune response in a subject, which, in some aspects,treats a disease or disorder, such as a cancer, in the subject. In someaspects, a PD-1-binding polypeptide provided herein, such as a PD-1-Fc,can bind to PD-1-expressing cells and block their interaction with PD-L1and/or PD-L2 on adjacent cells in the immune synapse, e.g. tumor cells,which, in some aspects, can induce an active immune response in theenvironment. In some cases, the active immune response can inhibit thegrowth (e.g., block cells cycle progression) of the cancerous cells.

In other aspects, also provided herein are VHH-binding polypeptides thatexhibit multispecific binding. In some cases, the binding polypeptidesinclude polypeptides that exhibit dual affinity for PD-1 and a tumorassociated antigen (TAA). Alternatively or additionally, PD-1 bindingpolypeptides include polypeptides that exhibit affinity for PD-1 and a Tcell antigen, such as CD3. In some aspects, such multispecific moleculesare capable of engaging or activating T cells at the site of a tumorupon binding to a tumor or T cell and simultaneously blockinginteractions of PD-1 and PD-L1/PD-L2 to reduce an inhibitory signal inthe T cells. In particular, among such molecules provided herein aremolecules that exhibit constrained CD3 binding. Also provided herein areengineered cells, such as engineered T cells, that express a chimericantigen receptor and that are capable of secreting a PD-1 bindingpolypeptide.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993); and updated versions thereof.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context or expressly indicated, singularterms shall include pluralities and plural terms shall include thesingular. For any conflict in definitions between various sources orreferences, the definition provided herein will control.

It is understood that embodiments of the invention described hereininclude “consisting” and/or “consisting essentially of” embodiments. Asused herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. Use of the term “or” herein isnot meant to imply that alternatives are mutually exclusive.

In this application, the use of “or” means “and/or” unless expresslystated or understood by one skilled in the art. In the context of amultiple dependent claim, the use of “or” refers back to more than onepreceding independent or dependent claim.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide”may be used interchangeably, and refer to a polymer of nucleotides. Suchpolymers of nucleotides may contain natural and/or non-naturalnucleotides, and include, but are not limited to, DNA, RNA, and PNA.“Nucleic acid sequence” refers to the linear sequence of nucleotidescomprised in the nucleic acid molecule or polynucleotide.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin (1) is not associated with all ora portion of a polynucleotide found in nature, (2) is operably linked toa polynucleotide that it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present disclosure, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldtypically be found in nature, (2) is essentially free of other proteinsfrom the same source, e.g., from the same species, (3) is expressed by acell from a different species, (4) has been separated from at leastabout 50 percent of polynucleotides, lipids, carbohydrates, or othermaterials with which it is associated in nature, (5) is not associated(by covalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of may be of synthetic origin, or any combinationthereof. In certain embodiments, the isolated protein is substantiallypure or substantially free from proteins or polypeptides or othercontaminants that are found in its natural environment that wouldinterfere with its use (therapeutic, diagnostic, prophylactic, researchor otherwise).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, for example, in someembodiments, more than about 85%, 90%, 95%, and 99%. In someembodiments, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “specifically binds” to an antigen or epitope is a term that iswell understood in the art, and methods to determine such specificbinding are also well known in the art. A molecule is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. A single-domain antibody (sdAb) orVHH-containing polypeptide “specifically binds” or “preferentiallybinds” to a target if it binds with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other substances.For example, a sdAb or VHH-containing polypeptide that specifically orpreferentially binds to a PD-1 epitope is a sdAb or VHH-containingpolypeptide that binds this epitope with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other PD-1epitopes or non-PD-1 epitopes. It is also understood by reading thisdefinition that; for example, a sdAb or VHH-containing polypeptide thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.“Specificity” refers to the ability of a binding protein to selectivelybind an antigen.

As used herein, the term “epitope” refers to a site on a target molecule(for example, an antigen, such as a protein, nucleic acid, carbohydrateor lipid) to which an antigen-binding molecule (for example, a sdAb orVHH-containing polypeptide) binds. Epitopes often include a chemicallyactive surface grouping of molecules such as amino acids, polypeptidesor sugar side chains and have specific three-dimensional structuralcharacteristics as well as specific charge characteristics. Epitopes canbe formed both from contiguous and/or juxtaposed noncontiguous residues(for example, amino acids, nucleotides, sugars, lipid moiety) of thetarget molecule. Epitopes formed from contiguous residues (for example,amino acids, nucleotides, sugars, lipid moiety) typically are retainedon exposure to denaturing solvents whereas epitopes formed by tertiaryfolding typically are lost on treatment with denaturing solvents. Anepitope may include but is not limited to at least 3, at least 5 or 8-10residues (for example, amino acids or nucleotides). In some embodiments,an epitope is less than 20 residues (for example, amino acids ornucleotides) in length, less than 15 residues or less than 12 residues.Two antibodies may bind the same epitope within an antigen if theyexhibit competitive binding for the antigen. In some embodiments, anepitope can be identified by a certain minimal distance to a CDR residueon the antigen-binding molecule. In some embodiments, an epitope can beidentified by the above distance, and further limited to those residuesinvolved in a bond (for example, a hydrogen bond) between a residue ofthe antigen-binding molecule and an antigen residue. An epitope can beidentified by various scans as well, for example an alanine or argininescan can indicate one or more residues that the antigen-binding moleculecan interact with. Unless explicitly denoted, a set of residues as anepitope does not exclude other residues from being part of the epitopefor a particular antigen-binding molecule. Rather, the presence of sucha set designates a minimal series (or set of species) of epitopes. Thus,in some embodiments, a set of residues identified as an epitopedesignates a minimal epitope of relevance for the antigen, rather thanan exclusive list of residues for an epitope on an antigen.

A “nonlinear epitope” or “conformational epitope” comprisesnoncontiguous polypeptides, amino acids and/or sugars within theantigenic protein to which an antigen-binding molecule specific to theepitope binds. In some embodiments, at least one of the residues will benoncontiguous with the other noted residues of the epitope; however, oneor more of the residues can also be contiguous with the other residues.

A “linear epitope” comprises contiguous polypeptides, amino acids and/orsugars within the antigenic protein to which an antigen-binding moleculespecific to the epitope binds. It is noted that, in some embodiments,not every one of the residues within the linear epitope need be directlybound (or involved in a bond) by the antigen-binding molecule. In someembodiments, linear epitopes can be from immunizations with a peptidethat effectively consisted of the sequence of the linear epitope, orfrom structural sections of a protein that are relatively isolated fromthe remainder of the protein (such that the antigen-binding molecule caninteract, at least primarily), just with that sequence section.

The terms “antibody” and “antigen-binding molecule” are usedinterchangeably in the broadest sense and encompass various polypeptidesthat comprise antibody-like antigen-binding domains, including but notlimited to conventional antibodies (typically comprising at least oneheavy chain and at least one light chain), single-domain antibodies(sdAbs, comprising just one chain, which is typically similar to a heavychain), VHH-containing polypeptides (polypeptides comprising at leastone heavy chain only antibody variable domain, or VHH), and fragments ofany of the foregoing so long as they exhibit the desired antigen-bindingactivity. In some embodiments, an antibody comprises a dimerizationdomain. Such dimerization domains include, but are not limited to, heavychain constant domains (comprising CH1, hinge, CH2, and CH3, where CH1typically pairs with a light chain constant domain, CL, while the hingemediates dimerization) and Fc domains (comprising hinge, CH2, and CH3,where the hinge mediates dimerization).

The term antibody also includes, but is not limited to, chimericantibodies, humanized antibodies, and antibodies of various species suchas camelid (including llama), shark, mouse, human, cynomolgus monkey,etc.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable regions of the heavy chain and lightchain (V_(H) and V_(L), respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three CDRs. (See, e.g., Kindt et al. KubyImmunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A singleV_(H) or V_(L) domain may be sufficient to confer antigen-bindingspecificity, e.g. a single domain antibody, such as a VHH. Furthermore,antibodies that bind a particular antigen may be isolated using a V_(H)or V_(L) domain from an antibody that binds the antigen to screen alibrary of complementary V_(L) or V_(H) domains, respectively. See,e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al.,Nature 352:624-628 (1991).

An “antibody fragment” or “antigen-binding fragment” refers to amolecule other than a conventional or intact antibody that comprises aportion of an conventional or intact antibody containing at least avariable region that binds an antigen. Examples of antibody fragmentsinclude but are not limited to Fv, single chain Fvs (sdFvs), Fab, Fab′,Fab′-SH, F(ab′)₂; diabodies; linear antibodies; an single-domainantibodies comprising only the V_(H) region (VHH).

As used herein, “monovalent” with reference to a binding molecule refersto binding molecules that have a single antigen recognition site that isspecific for a target antigen. Examples of monovalent binding moleculesinclude, for example, a monovalent antibody fragment, a proteinaceousbinding molecule with antibody-like binding properties or an MHCmolecule. Examples of monovalent antibody fragments include, but are notlimited to, a Fab fragment, an Fv fragment, and a single-chain Fvfragment (scFv).

The terms “single domain antibody”, “sdAb,” “VHH” are usedinterchangeably herein to refer to an antibody having a single monomericdomain antigen binding/recognition domain. Such antibodies include acamelid antibody or shark antibody. In some embodiments, a VHH comprisesthree CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2,FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at theN-terminus or C-terminus such that it comprise only a partial FR1 and/orFR4, or lacks one or both of those framework regions, so long as the VHHsubstantially maintains antigen binding and specificity.

The term “VHH-containing polypeptide” refers to a polypeptide thatcomprises at least one VHH domain. In some embodiments, a VHHpolypeptide comprises two, three, or four or more VHH domains, whereineach VHH domain may be the same or different. In some embodiments, aVHH-containing polypeptide comprises an Fc domain. In some suchembodiments, the VHH polypeptide may form a dimer. Nonlimitingstructures of VHH-containing polypeptides include VHH₁-Fc, VHH₁-VHH₂-Fc,and VHH₁-VHH₂-VHH₃-Fc, wherein VHH₁, VHH₂, and VHH₃ may be the same ordifferent. In some embodiments of such structures, one VHH may beconnected to another VHH by a linker, or one VHH may be connected to theFc by a linker. In some such embodiments, the linker comprises 1-20amino acids, preferably 1-20 amino acids predominantly composed ofglycine and, optionally, serine. In some embodiments, when aVHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, thestructure VHH₁-VHH₂-Fc, if it forms a dimer, is considered to betetravalent (i.e., the dimer has four VHH domains). Similarly, thestructure VHH₁-VHH₂-VHH₃-Fc, if it forms a dimer, is considered to behexavalent (i.e., the dimer has six VHH domains).

As used herein, a PD-1-binding polypeptide is a polypeptide or proteinthat specifically binds PD-1. Typically, a PD-1-binding polypeptideherein is a VHH-containing polypeptide containing at least one VHHdomain that binds PD-1. A PD-1-binding polypeptide includes conjugates,including fusion proteins. A PD-1-binding polypeptide includes fusionproteins, including those containing an Fc domain. In some embodiments,a PD-1-binding polypeptide contains two, three, or four or more VHHdomains that each specifically bind to PD-1, wherein each VHH domain maybe the same or different. In some embodiments, a PD-1-bindingpolypeptide is multivalent. In some embodiments, a PD-1-bindingpolypeptide is multispecific. In some cases, a PD-1-binding polypeptidemay contain one or more additional domains that bind to one or morefurther or additional antigens other than PD-1.

The term “monoclonal antibody” refers to an antibody (including an sdAbor VHH-containing polypeptide) of a substantially homogeneous populationof antibodies, that is, the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. Thus, a sample of monoclonalantibodies can bind to the same epitope on the antigen. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies may be made by thehybridoma method first described by Kohler and Milstein, 1975, Nature256:495, or may be made by recombinant DNA methods such as described inU.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al., 1990, Nature 348:552-554, for example.

The term “CDR” denotes a complementarity determining region as definedby at least one manner of identification to one of skill in the art. Theprecise amino acid sequence boundaries of a given CDR or FR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (1991), “Sequences of Proteinsof Immunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme);Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996),“Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme);Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev CompImmunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger Aand Pluckthun A, “Yet another numbering scheme for immunoglobulinvariable domains: an automatic modeling and analysis tool,” J Mol Biol,2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al.,“Modeling antibody hypervariable loops: a combined algorithm,” PNAS,1989, 86(23):9268-9272, (“AbM” numbering scheme).

The boundaries of a given CDR or FR may vary depending on the schemeused for identification. For example, the Kabat scheme is based onstructural alignments, while the Chothia scheme is based on structuralinformation. Numbering for both the Kabat and Chothia schemes is basedupon the most common antibody region sequence lengths, with insertionsaccommodated by insertion letters, for example, “30a,” and deletionsappearing in some antibodies. The two schemes place certain insertionsand deletions (“indels”) at different positions, resulting indifferential numbering. The Contact scheme is based on analysis ofcomplex crystal structures and is similar in many respects to theChothia numbering scheme. The AbM scheme is a compromise between Kabatand Chothia definitions based on that used by Oxford Molecular's AbMantibody modeling software.

In some embodiments, CDRs can be defined in accordance with any of theChothia numbering schemes, the Kabat numbering scheme, a combination ofKabat and Chothia, the AbM definition, and/or the contact definition. AVHH comprises three CDRs, designated CDR1, CDR2, and CDR3. Table 1,below, lists exemplary position boundaries of CDR-H1, CDR-H2, CDR-H3 asidentified by Kabat, Chothia, AbM, and Contact schemes, respectively.For CDR-H1, residue numbering is listed using both the Kabat and Chothianumbering schemes. FRs are located between CDRs, for example, with FR-H1located before CDR-H1, FR-H2 located between CDR-H1 and CDR-H2, FR-H3located between CDR-H2 and CDR-H3 and so forth. It is noted that becausethe shown Kabat numbering scheme places insertions at H35A and H35B, theend of the Chothia CDR-H1 loop when numbered using the shown Kabatnumbering convention varies between H32 and H34, depending on the lengthof the loop.

TABLE 1 Boundaries of CDRs according to various numbering schemes. CDRKabat Chothia AbM Contact CDR-H1 H31- - - H35B H26 - - - H32 . . . 34H26 - - - H35B H30 - - - H35B (Kabat Numbering¹) CDR-H1 H31- - - H35H26 - - - H32 H26 - - - H35 H30 - - - H35 (Chothia Numbering²) CDR-H2H50 - - - H65 H52 - - - H56 H50 - - - H58 H47 - - - H58 CDR-H3 H95 - - -H102 H95 - - - H102 H95 - - - H102 H93 - - - H101 ¹Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public HealthService, National Institutes of Health, Bethesda, MD ²Al-Lazikani etal., (1997) JMB 273, 927-948

Thus, unless otherwise specified, a “CDR” or “complementary determiningregion,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), ofa given antibody or region thereof, such as a variable region thereof,should be understood to encompass a (or the specific) complementarydetermining region as defined by any of the aforementioned schemes. Forexample, where it is stated that a particular CDR (e.g., a CDR-H3)contains the amino acid sequence of a corresponding CDR in a given VHHamino acid sequence, it is understood that such a CDR has a sequence ofthe corresponding CDR (e.g., CDR-H3) within the VHH, as defined by anyof the aforementioned schemes. In some embodiments, specific CDRsequences are specified. Exemplary CDR sequences of provided antibodiesare described using various numbering schemes (see e.g. Table 1),although it is understood that a provided antibody can include CDRs asdescribed according to any of the other aforementioned numbering schemesor other numbering schemes known to a skilled artisan.

As used herein, “conjugate,” “conjugation” or grammatical variationsthereof refers the joining or linking together of two or more compoundsresulting in the formation of another compound, by any joining orlinking methods known in the art. It can also refer to a compound whichis generated by the joining or linking together two or more compounds.For example, a VHH domain linked directly or indirectly to one or morechemical moieties or polypeptide is an exemplary conjugate. Suchconjugates include fusion proteins, those produced by chemicalconjugates and those produced by any other methods.

An immunoglobulin Fc fusion (“Fc-fusion”), such as VHH-Fc, is a moleculecomprising one or more VHH domains operably linked to an Fc region of animmunoglobulin. An immunoglobulin Fc region may be linked indirectly ordirectly to one or more VHH domains. Various linkers are known in theart and can optionally be used to link an Fc to a fusion partner togenerate an Fc-fusion. In some such embodiments, the linker comprises1-20 amino acids, preferably 1-20 amino acids predominantly composed ofglycine and, optionally, serine. Fc-fusions of identical species can bedimerized to form Fc-fusion homodimers, or using non-identical speciesto form Fc-fusion heterodimers. In some embodiments, the Fc is amammalian Fc such as human Fc.

The term “heavy chain constant region” as used herein refers to a regioncomprising at least three heavy chain constant domains, C_(H)1, hinge,C_(H)2, and C_(H)3. Of course, non-function-altering deletions andalterations within the domains are encompassed within the scope of theterm “heavy chain constant region,” unless designated otherwise.Nonlimiting exemplary heavy chain constant regions include γ, δ, and α.Nonlimiting exemplary heavy chain constant regions also include ε and μ.Each heavy constant region corresponds to an antibody isotype. Forexample, an antibody comprising a γ constant region is an IgG antibody,an antibody comprising a δ constant region is an IgD antibody, and anantibody comprising an α constant region is an IgA antibody. Further, anantibody comprising a μ constant region is an IgM antibody, and anantibody comprising an ε constant region is an IgE antibody. Certainisotypes can be further subdivided into subclasses. For example, IgGantibodies include, but are not limited to, IgG1 (comprising a γ₁constant region), IgG2 (comprising a γ₂ constant region), IgG3(comprising a γ₃ constant region), and IgG4 (comprising a γ₄ constantregion) antibodies; IgA antibodies include, but are not limited to, IgA1(comprising an α₁ constant region) and IgA2 (comprising an α₂ constantregion) antibodies; and IgM antibodies include, but are not limited to,IgM1 and IgM2.

A “Fc region” as used herein refers to a portion of a heavy chainconstant region comprising CH2 and CH3. In some embodiments, an Fcregion comprises a hinge, CH2, and CH3. In various embodiments, when anFc region comprises a hinge, the hinge mediates dimerization between twoFc-containing polypeptides. An Fc region may be of any antibody heavychain constant region isotype discussed herein. In some embodiments, anFc region is an IgG1, IgG2, IgG3, or IgG4.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include Fc receptorbinding; C1q binding and complement dependent cytotoxicity (CDC); Fcreceptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (for exampleB-cell receptor); and B-cell activation, etc. Such effector functionsgenerally require the Fc region to be combined with a binding domain(for example, an antibody variable domain) and can be assessed usingvarious assays.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. In some embodiments, a “variant Fc region” comprisesan amino acid sequence which differs from that of a native sequence Fcregion by virtue of at least one amino acid modification, yet retains atleast one effector function of the native sequence Fc region. In someembodiments, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, for example, from about one to about ten aminoacid substitutions, and preferably, from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. In some embodiments, the variant Fc regionherein will possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, atleast about 90% sequence identity therewith, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99% sequence identity therewith.

In general, the numbering of the residues in an immunoglobulin heavychain or portion thereof, such as an Fc region, is that of the EU indexas in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). The “EU index as in Kabat” refers to the residue numbering ofthe human IgG1 EU antibody.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcγR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (See, for example,Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, forexample, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. For example, theterm “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methodsof measuring binding to FcRn are known (see, for example, Ghetie andWard, Immunol. Today 18(12):592-598 (1997); Ghetie et al., NatureBiotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem.279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

An “acceptor human framework” as used herein is a framework comprisingthe amino acid sequence of a heavy chain variable domain (V_(H))framework derived from a human immunoglobulin framework or a humanconsensus framework, as discussed herein. An acceptor human frameworkderived from a human immunoglobulin framework or a human consensusframework can comprise the same amino acid sequence thereof, or it cancontain amino acid sequence changes. In some embodiments, the number ofamino acid changes are fewer than 10, or fewer than 9, or fewer than 8,or fewer than 7, or fewer than 6, or fewer than 5, or fewer than 4, orfewer than 3, across all of the human frameworks in a single antigenbinding domain, such as a VHH.

As used herein, a “chimeric antigen receptor” or “CAR” refers to anengineered receptor, which introduces an antigen specificity, via anantigen binding domain, onto cells to which it is engineered (forexample T cells such as naive T cells, central memory T cells, effectormemory T cells or combination thereof) thus combining the antigenbinding properties of the antigen binding domain with the T cellactivity (e.g. lytic capacity and self renewal) of T cells. A CARtypically includes an extracellular antigen-binding domain (ectodomain),a transmembrane domain and an intracellular signaling domain. Theintracellular signaling domain generally contains at least one ITAMsignaling domain, e.g. derived from CD3zeta, and optionally at least onecostimulatory signaling domain, e.g. derived from CD28 or 4-1BB.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (for example,an antibody or VHH-containing polypeptide) and its binding partner (forexample, an antigen). The affinity or the apparent affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (K_(D)) or the K_(D-apparent), respectively.Affinity can be measured by common methods known in the art (such as,for example, ELISA K_(D), KinExA, flow cytometry, and/or surface plasmonresonance devices), including those described herein. Such methodsinclude, but are not limited to, methods involving BIAcore®, Octet®, orflow cytometry.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antigen-binding molecule/antigen interaction. When theterm “K_(D)” is used herein, it includes K_(D) and K_(D-apparent).

In some embodiments, the K_(D) of the antigen-binding molecule ismeasured by flow cytometry using an antigen-expressing cell line andfitting the mean fluorescence measured at each antibody concentration toa non-linear one-site binding equation (Prism Software graphpad). Insome such embodiments, the K_(D) is K_(D-apparent).

The term “biological activity” refers to any one or more biologicalproperties of a molecule (whether present naturally as found in vivo, orprovided or enabled by recombinant means). Biological propertiesinclude, but are not limited to, binding a ligand, inducing orincreasing cell proliferation (such as T cell proliferation), andinducing or increasing expression of cytokines.

An “affinity matured” VHH-containing polypeptide refers to aVHH-containing polypeptide with one or more alterations in one or moreCDRs compared to a parent VHH-containing polypeptide that does notpossess such alterations, such alterations resulting in an improvementin the affinity of the VHH-containing polypeptide for antigen.

A “humanized VHH” as used herein refers to a VHH in which one or moreframework regions have been substantially replaced with human frameworkregions. In some instances, certain framework region (FR) residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, the humanized VHH can comprise residues that arefound neither in the original VHH nor in the human framework sequences,but are included to further refine and optimize VHH or VHH-containingpolypeptide performance. In some embodiments, a humanized VHH-containingpolypeptide comprises a human Fc region. As will be appreciated, ahumanized sequence can be identified by its primary sequence and doesnot necessarily denote the process by which the antibody was created.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between two ormore numeric values such that one of skill in the art would consider thedifference between the two or more values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said value. In some embodimentsthe two or more substantially similar values differ by no more thanabout any one of 5%, 10%, 15%, 20%, 25%, or 50%.

A polypeptide “variant” means a biologically active polypeptide havingat least about 80% amino acid sequence identity with the native sequencepolypeptide after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Such variants include, for instance, polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the polypeptide. In some embodiments, a variant will haveat least about 80% amino acid sequence identity. In some embodiments, avariant will have at least about 90% amino acid sequence identity. Insome embodiments, a variant will have at least about 95% amino acidsequence identity with the native sequence polypeptide.

As used herein, “percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequenceare defined as the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in the specificpeptide or polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

An amino acid substitution may include but are not limited to thereplacement of one amino acid in a polypeptide with another amino acid.Exemplary substitutions are shown in Table 2. Amino acid substitutionsmay be introduced into an antibody of interest and the products screenedfor a desired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 2 Original Residue Exemplary Substitutions Ala (A) Val; Leu; IleArg (R) Lys; Gln; Asn Asn (N) Gln; His; Asp, Lys; Arg Asp (D) Glu; AsnCys (C) Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H)Asn; Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L)Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu;Phe; Ile Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) ThrThr (T) Val; Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V)Ile; Leu; Met; Phe; Ala; Norleucine

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “vector” is used to describe a polynucleotide that can beengineered to contain a cloned polynucleotide or polynucleotides thatcan be propagated in a host cell. A vector can include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that can be used in colorimetric assays, forexample, β-galactosidase). The term “expression vector” refers to avector that is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E,CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include progeny ofa single host cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic DNA complement) to the originalparent cell due to natural, accidental, or deliberate mutation. A hostcell includes cells transfected in vivo with a polynucleotide(s) aprovided herein.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature or produced. For example, a polypeptide isreferred to as “isolated” when it is separated from at least some of thecomponents of the cell in which it was produced. Where a polypeptide issecreted by a cell after expression, physically separating thesupernatant containing the polypeptide from the cell that produced it isconsidered to be “isolating” the polypeptide. Similarly, apolynucleotide is referred to as “isolated” when it is not part of thelarger polynucleotide (such as, for example, genomic DNA ormitochondrial DNA, in the case of a DNA polynucleotide) in which it istypically found in nature, or is separated from at least some of thecomponents of the cell in which it was produced, for example, in thecase of an RNA polynucleotide. Thus, a DNA polynucleotide that iscontained in a vector inside a host cell may be referred to as“isolated”.

The terms “individual” and “subject” are used interchangeably herein torefer to an animal; for example a mammal. The term patient includeshuman and veterinary subjects. In some embodiments, methods of treatingmammals, including, but not limited to, humans, rodents, simians,felines, canines, equines, bovines, porcines, ovines, caprines,mammalian laboratory animals, mammalian farm animals, mammalian sportanimals, and mammalian pets, are provided. The subject can be male orfemale and can be any suitable age, including infant, juvenile,adolescent, adult, and geriatric subjects. In some examples, an“individual” or “subject” refers to an individual or subject in need oftreatment for a disease or disorder. In some embodiments, the subject toreceive the treatment can be a patient, designating the fact that thesubject has been identified as having a disorder of relevance to thetreatment, or being at adequate risk of contracting the disorder. Inparticular embodiments, the subject is a human, such as a human patient.

A “disease” or “disorder” as used herein refers to a condition wheretreatment is needed and/or desired.

The term “tumor cell”, “cancer cell”, “cancer”, “tumor”, and/or“neoplasm”, unless otherwise designated, are used herein interchangeablyand refer to a cell (or cells) exhibiting an uncontrolled growth and/orabnormal increased cell survival and/or inhibition of apoptosis whichinterferes with the normal functioning of bodily organs and systems.Included in this definition are benign and malignant cancers, polyps,hyperplasia, as well as dormant tumors or micrometastases.

The terms “cancer” and “tumor” encompass solid andhematological/lymphatic cancers and also encompass malignant,pre-malignant, and benign growth, such as dysplasia. Also, included inthis definition are cells having abnormal proliferation that is notimpeded (e.g. immune evasion and immune escape mechanisms) by the immunesystem (e.g. virus infected cells). Exemplary cancers include, but arenot limited to: basal cell carcinoma, biliary tract cancer; bladdercancer; bone cancer; brain and central nervous system cancer; breastcancer; cancer of the peritoneum; cervical cancer; choriocarcinoma;colon and rectum cancer; connective tissue cancer; cancer of thedigestive system; endometrial cancer; esophageal cancer; eye cancer;cancer of the head and neck; gastric cancer (including gastrointestinalcancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as wellas B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),and Meigs' syndrome.

The term “non-tumor cell” as used herein refers to a normal cells ortissue. Exemplary non-tumor cells include, but are not limited to:T-cells, B-cells, natural killer (NK) cells, natural killer T (NKT)cells, dendritic cells, monocytes, macrophages, epithelial cells,fibroblasts, hepatocytes, interstitial kidney cells, fibroblast-likesynoviocytes, osteoblasts, and cells located in the breast, skeletalmuscle, pancreas, stomach, ovary, small intestines, placenta, uterus,testis, kidney, lung, heart, brain, liver, prostate, colon, lymphoidorgans, bone, and bone-derived mesenchymal stem cells. The term “a cellor tissue located in the periphery” as used herein refers to non-tumorcells not located near tumor cells and/or within the tumormicroenvironment.

The term “cells or tissue within the tumor microenvironment” as usedherein refers to the cells, molecules, extracellular matrix and/or bloodvessels that surround and/or feed a tumor cell. Exemplary cells ortissue within the tumor microenvironment include, but are not limitedto: tumor vasculature; tumor-infiltrating lymphocytes; fibroblastreticular cells; endothelial progenitor cells (EPC); cancer-associatedfibroblasts; pericytes; other stromal cells; components of theextracellular matrix (ECM); dendritic cells; antigen presenting cells;T-cells; regulatory T-cells (Treg cells); macrophages; neutrophils;myeloid-derived suppressor cells (MDSCs) and other immune cells locatedproximal to a tumor. Methods for identifying tumor cells, and/orcells/tissues located within the tumor microenvironment are well knownin the art, as described herein, below.

In some embodiments, an “increase” or “decrease” refers to astatistically significant increase or decrease, respectively. As will beclear to the skilled person, “modulating” can also involve effecting achange (which can either be an increase or a decrease) in affinity,avidity, specificity and/or selectivity of a target or antigen, for oneor more of its ligands, binding partners, partners for association intoa homomultimeric or heteromultimeric form, or substrates; effecting achange (which can either be an increase or a decrease) in thesensitivity of the target or antigen for one or more conditions in themedium or surroundings in which the target or antigen is present (suchas pH, ion strength, the presence of co-factors, etc.); and/or cellularproliferation or cytokine production, compared to the same conditionsbut without the presence of a test agent. This can be determined in anysuitable manner and/or using any suitable assay known per se ordescribed herein, depending on the target involved.

As used herein, “an immune response” is meant to encompass cellularand/or humoral immune responses that are sufficient to inhibit orprevent onset or ameliorate the symptoms of disease (for example, canceror cancer metastasis). “An immune response” can encompass aspects ofboth the innate and adaptive immune systems.

As used herein, the terms “treating,” “treatment,” or “therapy” of adisease, disorder or condition is an approach for obtaining beneficialor desired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a human. For purposes of this disclosure, beneficial ordesired clinical results include, but are not limited to, any one ormore of: alleviation of one or more symptoms, diminishment of extent ofdisease, preventing or delaying spread (for example, metastasis, forexample metastasis to the lung or to the lymph node) of disease,preventing or delaying recurrence of disease, delay or slowing ofdisease progression, amelioration of the disease state, inhibiting thedisease or progression of the disease, inhibiting or slowing the diseaseor its progression, arresting its development, and remission (whetherpartial or total). Also encompassed by “treatment” is a reduction ofpathological consequence of a proliferative disease. The methodsprovided herein contemplate any one or more of these aspects oftreatment. In-line with the above, the term treatment does not requireone-hundred percent removal of all aspects of the disorder.

As used herein in the context of cancer, the terms “treatment” or,“inhibit,” “inhibiting” or “inhibition” of cancer refers to at least oneof: a statistically significant decrease in the rate of tumor growth, acessation of tumor growth, or a reduction in the size, mass, metabolicactivity, or volume of the tumor, as measured by standard criteria suchas, but not limited to, the Response Evaluation Criteria for SolidTumors (RECIST), or a statistically significant increase in progressionfree survival (PFS) or overall survival (OS).

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering a therapeutic agent. “Ameliorating”also includes shortening or reduction in duration of a symptom.

“Preventing,” “prophylaxis,” or “prevention” of a disease or disorderrefers to administration of a pharmaceutical composition, either aloneor in combination with another compound, to prevent the occurrence oronset of a disease or disorder or some or all of the symptoms of adisease or disorder or to lessen the likelihood of the onset of adisease or disorder.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. To “reduce” or“inhibit” is to decrease, reduce or arrest an activity, function, and/oramount as compared to a reference. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause an overall decrease of 10% orgreater. In some embodiments, by “reduce” or “inhibit” is meant theability to cause an overall decrease of 50% or greater. In someembodiments, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments,the amount noted above is inhibited or decreased over a period of time,relative to a control over the same period of time.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. Unless otherwise specified, the terms “reduce”, “inhibit”,or “prevent” do not denote or require complete prevention over all time,but just over the time period being measured.

The term “anti-cancer agent” is used herein in its broadest sense torefer to agents that are used in the treatment of one or more cancers.Exemplary classes of such agents in include, but are not limited to,chemotherapeutic agents, anti-cancer biologics (such as cytokines,receptor extracellular domain-Fc fusions, and antibodies), radiationtherapy, CAR-T therapy, therapeutic oligonucleotides (such as antisenseoligonucleotides and siRNAs) and oncolytic viruses.

The term “biological sample” means a quantity of a substance from aliving thing or formerly living thing. Such substances include, but arenot limited to, blood, (for example, whole blood), plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

The term “control” or “reference” refers to a composition known to notcontain an analyte (“negative control”) or to contain an analyte(“positive control”). A positive control can comprise a knownconcentration of analyte.

The terms “effective amount” or “therapeutically effective amount” referto a quantity and/or concentration of a composition containing an activeingredient (e.g. sdAb or VHH-containing polypeptide) that whenadministered into a patient either alone (i.e., as a monotherapy) or incombination with additional therapeutic agents, yields a statisticallysignificant decrease in disease progression as, for example, byameliorating or eliminating symptoms and/or the cause of the disease. Aneffective amount may be an amount that relieves, lessens, or alleviatesat least one symptom or biological response or effect associated with adisease or disorder, prevents progression of the disease or disorder, orimproves physical functioning of the patient. A therapeuticallyeffective amount of a composition containing an active agent may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the active agent to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the active agent areoutweighed by the therapeutically beneficial effects. A therapeuticallyeffective amount may be delivered in one or more administrations. Atherapeutically effective amount refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic and/or prophylactic result.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to a preparation which is in such form as to permit the biologicalactivity of the active ingredient(s) to be effective, and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. Hence, it is a compositionsuitable for pharmaceutical use in a mammalian subject, often a human. Apharmaceutical composition typically comprises an effective amount of anactive agent (e.g., sdAb or VHH-containing polypeptide) and a carrier,excipient, or diluent. The carrier, excipient, or diluent is typically apharmaceutically acceptable carrier, excipient or diluent, respectively.Such formulations may be sterile.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand are compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and sequential administrationin any order.

The term “concurrently” is used herein to refer to administration of twoor more therapeutic agents, where at least part of the administrationoverlaps in time, or where the administration of one therapeutic agentfalls within a short period of time relative to administration of theother therapeutic agent, or wherein the therapeutic effect of bothagents overlap for at least a period of time.

The term “sequentially” is used herein to refer to administration of twoor more therapeutic agents that does not overlap in time, or wherein thetherapeutic effects of the agents do not overlap.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contain dications and/or warnings concerning theuse of such therapeutic products.

An “article of manufacture” is any manufacture (for example, a packageor container) or kit comprising at least one reagent, for example, amedicament for treatment of a disease or disorder (for example, cancer),or a probe for specifically detecting a biomarker described herein. Insome embodiments, the manufacture or kit is promoted, distributed, orsold as a unit for performing the methods described herein.

The terms “label” and “detectable label” mean a moiety attached, forexample, to an antibody or antigen to render a reaction (for example,binding) between the members of the specific binding pair, detectable.The labeled member of the specific binding pair is referred to as“detectably labeled.” Thus, the term “labeled binding protein” refers toa protein with a label incorporated that provides for the identificationof the binding protein. In some embodiments, the label is a detectablemarker that can produce a signal that is detectable by visual orinstrumental means, for example, incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(for example, ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ³¹¹I, ¹⁷⁷Lu, ¹⁶⁶Ho,or ¹⁵³Sm); chromogens, fluorescent labels (for example, FITC, rhodamine,lanthanide phosphors), enzymatic labels (for example, horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (for example, leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags);and magnetic agents, such as gadolinium chelates. Representativeexamples of labels commonly employed for immunoassays include moietiesthat produce light, for example, acridinium compounds, and moieties thatproduce fluorescence, for example, fluorescein. In this regard, themoiety itself may not be detectably labeled but may become detectableupon reaction with yet another moiety.

II. VHH Domains Binding PD-1

Provided herein are PD-1-binding polypeptides that are VHH-containingpolypeptides containing at least one VHH domain that specifically bindsto PD-1. In some embodiments, the VHH domain binds human PD-1. In someembodiments, the VHH domain binds cynomolgus PD-1. In some embodiments,the VHH domain binds murine PD-1. In some embodiments, theVHH-containing polypeptides incorporate multiple copies of a VHH domainprovided herein. In such embodiments, the VHH-containing polypeptide mayincorporate multiple copies of the same VHH domain. In some embodiments,the VHH-containing polypeptides may incorporate multiple copies of a VHHdomain that are different but that recognize the same epitope on PD-1.The VHH-containing polypeptides can be formatted in a variety offormats, including any as described in Section III below.

A VHH domain is an antibody fragment that is a single monomeric variableantibody domain that is able to bind selectively to a specific antigen.With a molecular weight of only 12-15 kDa, VHH domains (also calledsingle-domain antibodies) are much smaller than common antibodies(150-160 kDa) which are composed of two heavy protein chains and twolight chains, and even smaller than Fab fragments (˜50 kDa, one lightchain and half a heavy chain) and single-chain variable fragments (˜25kDa, two variable domains, one from a light and one from a heavy chain).

Single domain antibodies are antibodies whose complementary determiningregions are part of a single domain polypeptide. Examples include, butare not limited to, heavy chain antibodies, antibodies naturally devoidof light chains, single domain antibodies derived from conventional4-chain antibodies, engineered antibodies and single domain scaffoldsother than those derived from antibodies. Single domain antibodies maybe derived from any species including, but not limited to mouse, human,camel, llama, alpaca, vicuna, guanaco, shark, goat, rabbit, and/orbovine. In some embodiments, a single domain antibody as used herein isa naturally occurring single domain antibody known as heavy chainantibody devoid of light chains. For clarity reasons, this variabledomain derived from a heavy chain antibody naturally devoid of lightchain is known herein as a VHH to distinguish it from the conventionalVH of four chain immunoglobulins. Such a VHH molecule can be derivedfrom antibodies raised in Camelidae species, for example in camel,llama, dromedary, alpaca, vicuna and guanaco. Other species besidesCamelidae may produce heavy chain antibodies naturally devoid of lightchain; such VHHs are within the scope of the disclosure.

Methods for the screening of VHH domains, including VHH-bindingpolypeptides, that possess the desired specificity for PD-1 include, butare not limited to, enzyme linked immunosorbent assay (ELISA), enzymaticassays, flow cytometry, and other immunologically mediated techniquesknown within the art.

Among the provided VHH domains provided herein are PD-1 VHH (llama oralpaca-derived) and humanized sequences, such as any described below.

In some embodiments, a VHH domain that binds PD-1 may be humanized.Humanized antibodies (such as VHH-containing polypeptides) are useful astherapeutic molecules because humanized antibodies reduce or eliminatethe human immune response to non-human antibodies, which can result inan immune response to an antibody therapeutic, and decreasedeffectiveness of the therapeutic. Generally, a humanized antibodycomprises one or more variable domains in which CDRs, (or portionsthereof) are derived from a non-human antibody, and FRs (or portionsthereof) are derived from human antibody sequences. A humanized antibodyoptionally will also comprise at least a portion of a human constantregion. In some embodiments, some FR residues in a humanized antibodyare substituted with corresponding residues from a non-human antibody(for example, the antibody from which the CDR residues are derived), forexample, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633,and are further described, for example, in Riechmann et al., (1988)Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol.Immunol. 28:489-498 (describing “resurfacing”); Dall'Acqua et al.,(2005) Methods 36:43-60 (describing “FR shuffling”); and Osbourn et al.,(2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer,83:252-260 (describing the “guided selection” approach to FR shuffling).

Human framework regions that can be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, for example, Sims et al. (1993) J. Immunol. 151:2296);framework regions derived from the consensus sequence of humanantibodies of a particular subgroup of heavy chain variable regions(see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA,89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature(somatically mutated) framework regions or human germline frameworkregions (see, for example, Almagro and Fransson, (2008) Front. Biosci.13:1619-1633); and framework regions derived from screening FR libraries(see, for example, Baca et al., (1997) J. Biol. Chem. 272: 10678-10684and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, theFR regions of a VHH are replaced with human FR regions to make ahumanized VHH. In some embodiments, certain FR residues of the human FRare replaced in order to improve one or more properties of the humanizedVHH. VHH domains with such replaced residues are still referred toherein as “humanized.”

Provided herein is a VHH domain that binds PD-1 comprising a CDR1, CDR2and CDR3 contained in a VHH amino acid sequences selected from any ofSEQ ID NO: 251-267, or 284, or an amino acid sequence that has at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe VHH region amino acid selected from any one of SEQ ID NOs: 251-267or 284.

In some embodiments, a PD-1 VHH domain provided herein contains a CDR1,CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO:284, or anamino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to the VHH region amino acid selectedset forth in SEQ ID NO: 284. In some embodiments, the PD-1 VHH domainhas the amino acid sequence set forth in SEQ ID NO: 284 or an amino acidsequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% sequence identity to the amino acid selected set forth in SEQ IDNO: 284. In some embodiments, the PD-1 VHH domain is a humanized variantof the amino acid sequence set forth in SEQ ID NO: 284.

In some embodiments, a PD-1 VHH domain provided herein contains a CDR1,CDR2, CDR3 contained in a VHH domain set forth in SEQ ID NO: 312, or anamino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to the VHH region amino acid selectedset forth in SEQ ID NO: 312. In some embodiments, the PD-1 VHH domainhas the amino acid sequence set forth in SEQ ID NO: 312 or an amino acidsequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% sequence identity to the amino acid selected set forth in SEQ IDNO: 312. In some embodiments, the PD-1 VHH domain is a humanized variantof the amino acid sequence set forth in SEQ ID NO: 312.

In some embodiments, a PD-1 VHH domain provided herein contains a CDR1set forth in any one of SEQ ID NOS: 268, 272, 273 or 313, a CDR2 setforth in SEQ ID NO: 278 or 314 and a CDR3 set forth in SEQ ID NO: 283 or315.

In some embodiments, the PD-1 VHH domain provided herein contains aCDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 272, 278, and 283,respectively. In some embodiments, the PD-1 VHH domain provided hereincontains a CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 268, 278, and283, respectively. In some embodiments, the PD-1 VHH domain providedherein contains a CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 272, 278,and 283, respectively. In some embodiments, the PD-1 VHH domain providedherein contains a CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 273, 278,and 283, respectively. In some embodiments, the PD-1 VHH domain providedherein contains a CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 313,314, and 315, respectively.

In some aspects, a VHH domain that binds PD-1 comprises a CDR1, CDR2 andCDR3 contained in a VHH amino acid sequences selected from any of SEQ IDNO:251-267, or an amino acid sequence that has at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the V_(H)Hregion amino acid selected from any one of SEQ ID NOs: 251-267.

In some cases, the provided PD-1 VHH domain is a humanized variant thathas the amino acid sequence set forth in any of SEQ ID NOS: 251-267 oran amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to the VHH region amino acidselected from any one of SEQ ID NOs: 251-267. In some embodiments, thePD-1 humanized VHH domain has the sequence of amino acids set forth inany one of SEQ ID NOS: 251-267.

Provided herein is a VHH domain that binds PD-1 comprising a CDR1, CDR2and CDR3 contained in a VHH amino acid sequences selected from any ofSEQ ID NO: 287, 288, 289, or 290, or an amino acid sequence that has atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the VHH region amino acid selected from any one of SEQ IDNOs: 296, 297, 298, or 299.

In some embodiments, the PD-1 VHH domain is a humanized variant of theamino acid sequence set forth in SEQ ID NO: 296, 297, 298, or 299.

In some embodiments, a PD-1 VHH domain provided herein contains a CDR1set forth in any one of SEQ ID NOS: 300, 303, 306, or 309, a CDR2 setforth in SEQ ID NO: 301, 304, 307, or 310, a CDR3 set forth in SEQ IDNO: 302, 305, 308, or 311. In some embodiments, a PD-1 VHH domainprovided herein contains a CDR1 set forth in SEQ ID NO: 300, a CDR2 setforth in SEQ ID NO: 301, and a CDR3 set forth in SEQ ID NO: 302; a PD-1VHH domain provided herein contains a CDR1 set forth in SEQ ID NO: 303,a CDR2 set forth in SEQ ID NO: 304, and a CDR3 set forth in SEQ ID NO:305; a PD-1 VHH domain provided herein contains a CDR1 of SEQ ID NO:306, a CDR2 set forth in SEQ ID NO: 307, and a CDR3 set forth in SEQ IDNO: 308; or a PD-1 VHH domain provided herein contains a CDR1 set forthin SEQ ID NO: 309, a CDR2 set forth in SEQ ID NO: 310, and a CDR3 setforth in SEQ ID NO: 311.

III. Fusion Proteins and Conjugates Containing PD-1-Binding Polypeptides

Provided herein are fusion proteins and conjugates containingPD-1-binding polypeptides containing at least one VHH domain thatspecifically binds PD-1 linked, directly or indirectly, to one or moreadditional domains or moieties. In some embodiments, the fusion proteinor conjugate of the present disclosure is composed of a singlepolypeptide. In other embodiments, the fusion protein or conjugate ofthe present disclosure is composed of more than one polypeptide. In someembodiments, the PD-1-binding polypeptide of the present disclosureincorporates at least one VHH domain that specifically binds PD-1. Insome aspects, the PD-1-binding polypeptide is multivalent. In someembodiments, the PD-1-binding polypeptides include two or more copies ofa VHH domain that specifically binds PD-1, for example, three or more,four or more, five or more, or six or more copies of a VHH domain thatspecifically binds PD-1. In certain aspects, the PD-1-bindingpolypeptide is multispecific. For example, in some cases, the one ormore additional domain may be one or more additional binding domain thatbinds to one or more further antigen or protein.

In some embodiments, the PD-1-binding polypeptides of the presentdisclosure include two or more polypeptide sequences that are operablylinked via amino acid linkers. In some embodiments, these linkers arecomposed predominately of the amino acids Glycine and Serine, denoted asGS-linkers herein. The GS-linkers of the fusion proteins of the presentdisclosure can be of various lengths, for example, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. In someembodiments, the GS-linker comprises an amino acid sequence selectedfrom the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ ID NO: 1);GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 2); GGSGGSGGSGGS, i.e., (GGS)₄ (SEQID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 4). In someembodiments, the linker is a flexible linker comprising Glycineresidues, such as, by way of non-limiting example, GG, GGG, GGGG (SEQ IDNO: 5), GGGGG (SEQ ID NO: 6), and GGGGGG (SEQ ID NO: 7). In someembodiments, the PD-1-binding polypeptide includes a combination of aGS-linker and a Glycine linker.

A. Fc Fusions

Provided herein is a PD-1-binding polypeptide that is a fusion proteincontaining at least one VHH domain that binds PD-1 provided herein andan Fc domain. In some embodiments, a PD-1-binding polypeptide providedherein comprises one, two, three, or four VHH domains that bind PD-1 andan Fc domain.

In some embodiments, incorporation of an immunoglobulin Fc region intothe fusion protein can, in some aspects, be composed of two polypeptidesthat together form a dimer. In some embodiments, an Fc domain mediatesdimerization of the PD-1-binding polypeptide at physiologicalconditions, such as when expressed from a cell, such that a dimer isformed that doubles the number of PD-1 binding sites. For example, aPD-1-binding polypeptide comprising three VHH domains that bind PD-1 andan Fc region is trivalent as a monomer, but the Fc region may mediatedimerization, such that the PD-1-binding polypeptide exists as ahexavalent dimer under such conditions. In some embodiments, a PD-1 VHHdomain is fused to an IgG Fc region and in these embodiments, the fusionprotein is bivalent having two PD-1 VHH domains per molecule. In someembodiments, two PD-1 binding domains (2×) are fused to an IgG Fc regionand in these embodiments, the fusion protein is tetravalent having fourPD-1 VHH domains per molecule. In some embodiments, three PD-1 VHHdomain (3×) are fused to an IgG Fc region and in these embodiments, thefusion protein is hexavalent having six PD-1 VHH domains per molecule.

In some embodiments, the multivalent PD-1-binding polypeptide isbivalent. In some embodiments, the bivalent PD-1-binding polypeptide ofthe disclosure includes two copies of a PD-1-binding polypeptide havingthe following structure: (PD-1 VHH)-Linker-Fc. In some embodiments, themultivalent PD-1-binding polypeptide is tetravalent. In someembodiments, the tetravalent PD-1-binding polypeptide of the disclosureincludes two copies of a PD-1-polypeptide having the followingstructure: (PD-1 VHH)-Linker-(PD-1 VHH)-Linker-Fc. In some embodiments,the multivalent PD-1-binding polypeptide is hexavalent. In someembodiments, the hexavalent PD-1-binding polypeptide of the disclosureincludes two copies of a PD-1-binding polypeptide having the followingstructure: (PD-1 VHH)-Linker-(PD-1 VHH)-Linker-(PD-1 VHH)-Linker-Fc.

In some cases, the CH3 domain of the Fc region can be used ashomodimerization domain, such that the resulting fusion protein isformed from two identical polypeptides. In other cases, the CH3 dimerinterface region of the Fc region can be mutated so as to enableheterodimerization. For example, a heterodimerization domain can beincorporated into the fusion protein such that the construct is anasymmetric fusion protein.

In any of the provided embodiments, a PD-1 VHH domain can be any asdescribed above. In come embodiments, the PD-1 VHH domain is a humanizedVHH domain that binds PD-1.

In various embodiments, an Fc domain included in a PD-1-bindingpolypeptide is a human Fc domain, or is derived from a human Fc domain.In some embodiments, the fusion protein contains an immunoglobulin Fcregion. In some embodiments, the immunoglobulin Fc region is an IgGisotype selected from the group consisting of IgG1 isotype, IgG2isotype, IgG3 isotype, and IgG4 subclass.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof is an IgG isotype. For example, theimmunoglobulin Fc region of the fusion protein is of human IgG1 isotype,having an amino acid sequence:

(SEQ ID NO: 8) PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHEDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof comprises a human IgG1 polypeptide sequence thatis at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQID NO: 8.

In some embodiments where the fusion protein of the disclosure includesan Fc polypeptide, the Fc polypeptide is mutated or modified. In somecases, the mutations include one or more amino acid substitutions toreduce an effector function of the Fc polypeptide. Various examples ofmutations to Fc polypeptides to alter, such as reduce, effector functionare known, including any as described below. In some embodiments,reference to amino acid substitutions in an Fc region is by EU numberingby Kabat (also called Kabat numbering) unless described with referenceto a specific SEQ ID NO. EU numbering is known and is according to themost recently updated IMGT Scientific Chart (IMGT®, the internationalImMunoGeneTics information System®,http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html(created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index asreported in Kabat, E. A. et al. Sequences of Proteins of Immunologicalinterest. 5th ed. US Department of Health and Human Services, NIHpublication No. 91-3242 (1991).

In some embodiments, an Fc region that exhibits reduced effectorfunctions may be a desirable candidate for applications in which PD-1 orCD3 binding is desired yet certain effector functions (such as CDC andADCC) are unnecessary or deleterious. In vitro and/or in vivocytotoxicity assays can be conducted to confirm the reduction/depletionof CDC and/or ADCC activities. For example, Fc receptor (FcR) bindingassays can be conducted to ensure that the multispecific polypeptideconstructs and/or cleaved components thereof lack FcγR binding (hencelikely lacking ADCC activity), but retains FcRn binding ability. Theprimary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. Non-limitingexamples of in vitro assays to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom,I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom,I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No.5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assay methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the multispecific polypeptideconstruct or cleaved components thereof is unable to bind C1q and hencelacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

In some embodiments, the human IgG Fc region is modified to alterantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), e.g., the amino acidmodifications described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al.,2010 mAbs, 2(2): 181-189; Lazar et al., 2006 PNAS, 103(11): 4005-4010,Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007Cancer Res, 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. EnzymeRegul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468;Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

Examples of mutations that enhance ADCC include modification at Ser239and Ile332, for example Ser239Asp and Ile332Glu (S239D, I332E). Examplesof mutations that enhance CDC include modifications at Lys326 andGlu333. In some embodiments, the Fc region is modified at one or both ofthese positions, for example Lys326Ala and/or Glu333Ala (K326A andE333A) using the Kabat numbering system.

In some embodiments, the Fc region of the fusion protein is altered atone or more of the following positions to reduce Fc receptor binding:Leu 234 (L234), Leu235 (L235), Asp265 (D265), Asp270 (D270), Ser298(S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327) or Pro329 (P329).For example, Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E),Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn (D270N), Ser298Asn(S298N), Asn297Ala (N297A), Pro329Ala (P329A) or Pro239Gly (P329G),Asn325Glu (N325E) or Ala327Ser (A327S). In preferred embodiments,modifications within the Fc region reduce binding to Fc-receptor-gammareceptors while have minimal impact on binding to the neonatal Fcreceptor (FcRn).

In some embodiments, the human IgG1 Fc region is modified at amino acidAsn297 (Kabat Numbering) to prevent glycosylation of the fusion protein,e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, theFc region of the fusion protein is modified at amino acid Leu235 (KabatNumbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) orLeu235Ala (L235A). In some embodiments, the Fc region of the fusionprotein is modified at amino acid Leu234 (Kabat Numbering) to alter Fcreceptor interactions, e.g., Leu234Ala (L234A). In some embodiments, theFc region of the fusion protein is modified at amino acid Leu234 (KabatNumbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E).In some embodiments, the Fc region of the fusion protein is altered atboth amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala(L234A/L235A) or Leu234Val and Leu235Ala (L234V/L235A). In someembodiments, the Fc region of the fusion protein is altered at aminoacids at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala(L234A/L235A/N297A). In some embodiments, the Fc region of the fusionprotein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala,Leu235Ala, Pro239Ala (L234A/L235A/P329A). In some embodiments, the Fcregion of the fusion protein is modified at amino acid Asp265 (KabatNumbering) to alter Fc receptor interactions, e.g Asp265Ala (D265A). Insome embodiments, the Fc region of the fusion protein is modified atamino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions,e.g Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fcregion of the fusion protein is altered at both amino acids 265 and 329,e.g., Asp265Ala and Pro329Ala (D265A/P329A) or Asp265Ala and Pro329Gly(D265A/P329G). In some embodiments, the Fc region of the fusion proteinis altered at amino acids at 234, 235, and 265, e.g., Leu234Ala,Leu235Ala, Asp265Ala (L234A/L235A/D265A). In some embodiments, the Fcregion of the fusion protein is altered at amino acids at 234, 235, and329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A/L235A/P329G). In someembodiments, the Fc region of the fusion protein is altered at aminoacids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala,Pro329Gly (L234A/L235A/D265A/P329G). In some embodiments, the Fc regionof the fusion protein is altered at Gly235 to reduce Fc receptorbinding. For example, wherein Gly235 is deleted from the fusion protein.In some embodiments, the human IgG1 Fc region is modified at amino acidGly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A).In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index ofKabat et al 1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the Fc region of the fusion protein is lacking anamino acid at one or more of the following positions to reduce Fcreceptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). Insome embodiments, the Fc region of the fusion protein is lacking anamino acid at one or more of the following positions Glu233 (E233),Leu234 (L234), or Leu235 (L235) and is modified at one or more of theAsp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptorbinding. For example, an Fc region included in a PD-1-bindingpolypeptide is derived from a human Fc domain, and comprises a threeamino acid deletion in the lower hinge corresponding to IgG1 E233, L234,and L235. In some aspects, such Fc polypeptides do not engage FcγRs andthus are referred to as “effector silent” or “effector null.” Forexample, Fc deletion of these three amino acids reduces the complementprotein C1q binding. In some embodiments, a polypeptide with an Fcregion with Fc deletion of these three amino acids retains binding toFcRn and therefore has extended half-life and transcytosis associatedwith FcRn mediated recycling. Such a modified Fc region is referred toas “Fc xELL” or “Fc deletion” and has the following amino acid sequence:

(SEQ ID NO: 9) PAPGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLPPSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment thereof comprises a human IgG1 polypeptide sequence thatis at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQID NO: 9.

In some embodiments, the human IgG Fc region is modified to enhance FcRnbinding. Examples of Fc mutations that enhance binding to FcRn areMet252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively)(Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol. 281(33)23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al2010 Nature Biotech, Vol. 28(2) 157-159), or Met252Ile, Thr256Asp,Met428Leu (M252I, T256D, M428L, respectively), (EU index of Kabat et al1991 Sequences of Proteins of Immunological Interest).

In some embodiments, the Fc domain included in a PD-1-bindingpolypeptide is derived from a human Fc domain and comprises mutationsM252Y and M428V, herein referred to as “Fc-YV”. In some embodiments, themutated or modified Fc polypeptide includes the following mutations:M252Y and M428L using the Kabat numbering system. In some embodiments,such mutations enhance binding to FcRn at the acidic pH of the endosome(near 6.5), while losing detectable binding at neutral pH (about 7.2),allowing for enhanced FcRn mediated recycling and extended half-life.

In some embodiments, the Fc domain included in a PD-1-bindingpolypeptide is derived from a human Fc domain and comprises mutations toinduce heterodimerization. In some embodiments, such mutations includethose referred to as “knob” and “hole” mutations. For example, having anamino acid modification within the CH3 domain at Thr366, which whenreplaced with a more bulky amino acid, e.g., Try (T366W), is able topreferentially pair with a second CH3 domain having amino acidmodifications to less bulky amino acids at positions Thr366, Leu368, andTyr407, e.g., Ser, Ala and Val, respectively (T366S/L368A/Y407V). Insome embodiments, the “knob” Fc domain comprises the mutation T366W. Insome embodiments, the “hole” Fc domain comprises mutations T366S, L368A,and Y407V. Heterodimerization via CH3 modifications can be furtherstabilized by the introduction of a disulfide bond, for example bychanging Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3domains (Reviewed in Carter, 2001 Journal of Immunological Methods, 248:7-15). In some embodiments, Fc domains used for heterodimerizationcomprise additional mutations, such as the mutation S354C on a firstmember of a heterodimeric Fc pair that forms an asymmetric disulfidewith a corresponding mutation Y349C on the second member of aheterodimeric Fc pair. In some embodiments, one member of aheterodimeric Fc pair comprises the modification H435R or H435K toprevent protein A binding while maintaining FcRn binding. In someembodiments, one member of a heterodimeric Fc pair comprises themodification H435R or H435K, while the second member of theheterodimeric Fc pair is not modified at H435. In various embodiments,the hole Fc domain comprises the modification H435R or H435K (referredto as “hole-R” in some instances when the modification is H435R), whilethe knob Fc domain does not. In some instances, the hole-R mutationimproves purification of the heterodimer over homodimeric hole Fcdomains that may be present.

In some embodiments, the human IgG Fc region is modified to preventdimerization. In these embodiments, the fusion proteins of the presentdisclosure are monomeric. For example modification at residue Thr366 toa charged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu(T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3dimerization.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG2 isotype, havingan amino acid sequence:

(SEQ ID NO: 10) PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTF RVVSVLTVVHQDWLNGKEYK CKVSNKGLPA PIEKTISKTK GQPREPQVYTLPPSREEMTK NQVSLTCLVK GFYPSDISVE WESNGQPENNYKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

In some embodiments, the fusion or immunologically active fragmentthereof comprises a human IgG2 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the human IgG2 Fc region is modified at amino acidAsn297 (e.g. to prevent to glycosylation of the antibody, e.g.,Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the humanIgG2 Fc region is lacks Lys447 (EU index of Kabat et al 1991 Sequencesof Proteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG3 isotype, havingan amino acid sequence:

(SEQ ID NO: 11) PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHEDPEVQFKWYV DGVEVHNAKT KPREEQYNST FRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTISKT KGQPREPQVYTLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESSGQPENNYNTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNIFSCSVMH EALHNRFTQK SLSLSPGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG3 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the human IgG3 Fc region is modified at amino acidAsn297 (Kabat Numbering) to prevent to glycosylation of the antibody,e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, thehuman IgG3 Fc region is modified at amino acid 435 to extend thehalf-life, e.g., Arg435His (R435H). In some embodiments, the human IgG3Fc region is lacks Lys447 (EU index of Kabat et al 1991 Sequences ofProteins of Immunological Interest).

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG4 isotype, havingan amino acid sequence:

(SEQ ID NO: 12) PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQEDPEVQFNWYV DGVEVHNAKT KPREEQFNST YRVVSVLTVLHQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG4 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the immunoglobulin Fc region or immunologicallyactive fragment of the fusion protein is of human IgG4 isotype, havingan amino acid sequence:

(SEQ ID NO: 13) PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQEDPEVQFNWYV DGVEVHNAKT KPREEQFNST YRVVSVLTVLHQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK

In some embodiments, the antibody or immunologically active fragmentthereof comprises a human IgG4 polypeptide sequence that is at least50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the human IgG4 Fc region is modified at amino acid235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In someembodiments, the human IgG4 Fc region is modified at amino acid Asn297(Kabat Numbering) to prevent to glycosylation of the antibody, e.g.,Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the humanIgG4 Fc region is lacks Lys447 (EU index of Kabat et al 1991 Sequencesof Proteins of Immunological Interest).

In some embodiments, the fusion protein contains a polypeptide derivedfrom an immunoglobulin hinge region. The hinge region can be selectedfrom any of the human IgG subclasses. For example, the fusion proteinmay contain a modified IgG1 hinge having the sequence of EPKSSDKTHTCPPC(SEQ ID NO: 14), where in the Cys220 that forms a disulfide with theC-terminal cysteine of the light chain is mutated to serine, e.g.,Cys220Ser (C220S). In other embodiments, the fusion protein contains atruncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 15).

In some embodiments, the fusion protein has a modified hinge from IgG4,which is modified to prevent or reduce strand exchange, e.g., Ser228Pro(S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 16). In someembodiments, the fusion protein contains linker polypeptides. In otherembodiments, the fusion protein contains linker and hinge polypeptides.

In some embodiments, the Fc region lacks or has reduced Fucose attachedto the N-linked glycan-chain at N297. There are numerous ways to preventfucosylation, including but not limited to production in a FUT8deficient cell line; addition inhibitors to the mammalian cell culturemedia, for example Castanospermine; and metabolic engineering of theproduction cell line.

In some embodiments, the Fc region is engineered to eliminaterecognition by pre-existing antibodies found in humans. In someembodiments, VHH-containing polypeptides of the present disclosure aremodified by mutation of position Leu11, for example Leu11Glu (L11E) orLeu11Lys (L11K). In other embodiments, single domain antibodies of thepresent disclosure are modified by changes in carboxy-terminal region,for example the terminal sequence has the sequence GQGTLVTVKPGG (SEQ IDNO: 17) or GQGTLVTVEPGG (SEQ ID NO: 18) or modification thereof. In someembodiments, the VHH-containing polypeptides of the present disclosureare modified by mutation of position 11 and by changes incarboxy-terminal region.

In some embodiments, the one or more polypeptides of the fusion proteinsof the present disclosure are operably linked via amino acid linkers. Insome embodiments, these linkers are composed predominately of the aminoacids Glycine and Serine, denoted as GS-linkers herein. The GS-linkersof the fusion proteins of the present disclosure can be of variouslengths, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 amino acids in length.

In some embodiments, the GS-linker comprises an amino acid sequenceselected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ ID NO:1); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 2); GGSGGSGGSGGS, i.e., (GGS)₄(SEQ ID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 4). Insome embodiments, the linker is a flexible linker comprising Glycineresidues, such as, by way of non-limiting example, GG, GGG, GGGG (SEQ IDNO: 5), GGGGG (SEQ ID NO: 6), and GGGGGG (SEQ ID NO: 7). In someembodiments, the fusion proteins can include a combination of aGS-linker and a Glycine linker.

B. Conjugates

Provided herein are conjugates containing at least one VHH domain thatspecifically binds PD-1 provided herein and one or more further moiety.The further moiety can be a therapeutic agent, such as a cytotoxicagent, or can be a detection agent. In some embodiments, the moiety canbe a targeting moiety, a small molecule drug (non-polypeptide drug ofless than 500 Daltons molar mass), a toxin, a cytostatic agent, acytotoxic agent, an immunosuppressive agent, a radioactive agentsuitable for diagnostic purposes, a radioactive metal ion fortherapeutic purposes, a prodrug-activating enzyme, an agent thatincreases biological half-life, or a diagnostic or detectable agent.

In some embodiments, the conjugate is an antibody drug conjugate (ADC,also called immunoconjugates) containing one or more PD-1 VHH domainprovided herein conjugated to a therapeutic agent, which is eithercytotoxic, cytostatic or otherwise provides some therapeutic benefit. Insome embodiments, the cytotoxic agent is a chemotherapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). In someembodiments, provided antibody drug conjugates of the present disclosureallow targeted-delivery of the drug moiety to tumors. In some cases,this can result in targeted killing of the tumor cell.

In some embodiments, there is provided a PD-1-binding conjugatecomprising at least one PD-1 VHH domain provided herein conjugated witha therapeutic agent. In some embodiments, the therapeutic agentincludes, for example, daunomycin, doxorubicin, methotrexate, andvindesine (Rowland et al., Cancer Immunol. Immunother. 21:183-187,1986). In some embodiments, the therapeutic agent has an intracellularactivity. In some embodiments, the PD-1-binding conjugate isinternalized and the therapeutic agent is a cytotoxin that blocks theprotein synthesis of the cell, therein leading to cell death. In someembodiments, the therapeutic agent is a cytotoxin comprising apolypeptide having ribosome-inactivating activity including, forexample, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin,diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variantsthereof. In some embodiments, where the therapeutic agent is a cytotoxincomprising a polypeptide having a ribosome-inactivating activity, thePD-1-binding conjugate must be internalized upon binding to the targetcell in order for the protein to be cytotoxic to the cells.

In some embodiments, there is provided a PD-1-binding conjugatecomprising at least one PD-1 VHH domain provided herein conjugated witha toxin. In some embodiments, the toxin includes, for example, bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al., J. Nat. CancerInst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem.Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem.13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al.,Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342(1993)). The toxins may exert their cytotoxic and cytostatic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition.

In some embodiments, there is provided a PD-1-binding conjugatecomprising at least one PD-1 VHH domain provided herein conjugated witha label, which can generate a detectable signal, indirectly or directly.These IgSF conjugates can be used for research or diagnosticapplications, such as for the in vivo detection of cancer. The label ispreferably capable of producing, either directly or indirectly, adetectable signal. For example, the label may be radio-opaque or aradioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; a fluorescent(fluorophore) or chemiluminescent (chromophore) compound, such asfluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such asalkaline phosphatase, β-galactosidase or horseradish peroxidase; animaging agent; or a metal ion. In some embodiments, the label is aradioactive atom for scintigraphic studies, for example 99Tc or 123I, ora spin label for nuclear magnetic resonance (NMR) imaging (also known asmagnetic resonance imaging, MRI), such as zirconium-89, iodine-123,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron. Zirconium-89 may be complexed to variousmetal chelating agents and conjugated to antibodies, e.g., for PETimaging (WO 2011/056983).

The PD-1-binding conjugates may be prepared using any methods known inthe art. See, e.g., WO 2009/067800, WO 2011/133886, and U.S. PatentApplication Publication No. 2014322129, incorporated by reference hereinin their entirety.

In some embodiments, the attachment can be covalent or non-covalent,e.g., via a biotin-streptavidin non-covalent interaction. In someembodiments, 1, 2, 3, 4, 5 or more moieties, which can be the same ordifferent, are conjugated, linked or fused to a PD-1 VHH domain to forma PD-1-binding conjugate. In some embodiments, such moieties can beattached to the VHH domain using various molecular biological orchemical conjugation and linkage methods known in the art and describedbelow. In some embodiments, linkers such as peptide linkers, cleavablelinkers, non-cleavable linkers or linkers that aid in the conjugationreaction, can be used to link or conjugate the effector moieties to thevariant polypeptide or immunomodulatory protein.

In some embodiments, a PD-1 VHH domain is conjugated to one or moremoieties, e.g. about 1 to about 20 drug moieties per VHH, through alinker (L). In some embodiments, the PD-1-binding conjugate comprisesthe following components: (VHH domain), (L)_(q) and (moiety)_(m),wherein the VHH domain is any of the described VHH domains capable ofspecifically binding PD-1 as described; L is a linker for linking theprotein or polypeptide to the moiety; m is at least 1; q is 0 or more;and the resulting PD-1-binding conjugate binds to PD-1. In particularembodiments, m is 1 to 4 and q is 0 to 8.

The linker may be composed of one or more linker components. Forcovalent attachment of the antibody and the drug moiety the linkertypically has two reactive functional groups, i.e. bivalency in areactive sense. Bivalent linker reagents which are useful to attach twoor more functional or biologically active moieties, such as peptides,nucleic acids, drugs, toxins, antibodies, haptens, and reporter groupsare known, and methods have been described their resulting conjugates(Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: NewYork, p 234-242).

Exemplary linker components include 6-maleimidocaproyl (“MC”),maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”), aalanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”),N-Succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-I carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”).

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, at a plasmin protease.

Conjugates of a VHH domain and cytotoxic agent can be made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl substrate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

The antibody drug conjugate can be prepared by a variety of methods,such as organic chemistry reactions, conditions, and reagents known tothose skilled in the art. In one embodiments, methods include: (1)reaction of a nucleophilic group of a VHH domain with a bivalent linkerreagent, to form VHH-L, via a covalent bond, followed by reaction with adrug moiety D; and (2) reaction of a nucleophilic group of a drug moietywith a bivalent linker reagent, to form D-L, via a covalent bond,followed by reaction with the nucleophilic group of a VHH domain.

Nucleophilic groups on antibodies, including VHH domains, include, butare not limited to: (i) N-terminal amine groups, (ii) side chain aminegroups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and(iv) sugar hydroxyl or amino groups where the antibody is glycosylated.Amine, thiol, and hydroxyl groups are nucleophilic and capable ofreacting to form covalent bonds with electrophilic groups on linkermoieties and linker reagents including: (i) active esters such as NHSesters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Conjugates, such as antibody drug conjugates, may also be produced bymodification of an antibody, such as a VHH domain, to introduceelectrophilic moieties, which can react with nucleophilic substituentson the linker reagent or drug. The sugars of glycosylated antibodies maybe oxidized, e.g., with periodate oxidizing reagents, to form aldehydeor ketone groups which may lead with the amine group of linker reagentsor drug moieties. The resulting imine Schiff base groups may form astable linkage, or may be reduced, e.g., by borohydride reagents to formstable amine linkages. In one embodiment, reaction of the carbohydrateportion of a glycosylated antibody with either galactose oxidase orsodium meta-periodate may yield carbonyl (aldehyde and ketone) groups inthe protein that can react with appropriate groups on the drug(Hermanson, Bioconjugate Techniques). In another embodiment, proteinscontaining N-terminal serine or threonine residues can react with sodiummeta-periodate, resulting in production of an aldehyde in place of thefirst amino acid. Such aldehyde can be reacted with a drug moiety orlinker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBi esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein containing a VHH domain and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

C. Multispecific Formats

Provided herein are PD-1-binding polypeptides that are multispecificcontaining at least one VHH domain that binds PD-1 and one or moreadditional binding domains. Typically, the one or more additionaldomains bind to a second antigen or protein other than PD-1. In someaspects, the further antigen or protein may be an antigen expressed on atumor, a molecule or receptor expressed on an immune cell, such as a Tcell, e.g. a CD3, or an additional inhibitory receptor (e.g. CTLA-4,LAG3, TIM3, VISTA, TIGIT, SIRPα, NKG2A, B7H3, B7H4) or an activatingreceptor (e.g. OX40, GITR, 41BB, CD40, CD27, CD28 or ICOS) or to conferan additional specificity to a target cell (e.g. CD8 or CD4). In someembodiments, the one or more additional domain is an antibody orantigen-binding fragment specific for the second antigen or protein. Insome embodiments, the additional domain is a VHH domain.

In some embodiments, a multispecific PD-1-binding polypeptide comprisesat least one VHH domain that binds PD-1 and at least one additionalbinding domain that binds a second antigen or protein. In someembodiments, this second antigen is a tumor associated antigen (TAA) ortumor microenvironment associated antigen (TMEAA). In some embodiments,this second antigen is an immunomodulatory antigen, wherein said antigenis involved with enhancing or dampening a signaling pathway in an immunecell.

In some cases, a multispecific PD-1-binding polypeptide can furthercontain an Fc domain, such as any described above. In some embodiments,a multispecific PD-1-binding polypeptide provided herein at least oneVHH domains that bind PD-1, at least one additional binding domain thatbinds a second antigen or protein, and an Fc domain. In someembodiments, an Fc domain mediates dimerization of the multispecificPD-1-binding polypeptide at physiological conditions such that a dimeris formed that doubles the number of binding sites for PD-1 and for theadditional antigen or protein.

Non-limiting exemplary multispecific PD-1-binding polypeptides aredescribed below.

1. Bispecific T Cells Engager

In some embodiments, the PD-1-binding polypeptide is a bispecificconstruct that is or comprises at least one PD-1 VHH domain providedherein and at least one additional binding molecule capable of bindingto a surface molecule expressed on a T cell. In some embodiments, thesurface molecule is an activating component of a T cell, such as acomponent of the T cell receptor complex. In particular aspects, thesurface molecule is an activating T cell antigen that is expressed on aT cell and is capable of inducing T cell activation upon interactionwith an antigen binding molecule. For example, in some aspects,interaction of an antigen binding molecule with an activating T cellantigen may induce T cell activation by triggering the signaling cascadeof the T cell receptor complex. Suitable assays to measure T cellactivation are known, and include any assay to measure or assessproliferation, differentiation, cytokine secretion, cytotoxic activityand/or expression of one or more activation marker. In some embodiments,the simultaneous or near simultaneous binding of such a PD-1-bindingpolypeptide to both of its targets, PD-1 expressed on target cell and aT cell molecule expressed on a T cell, e.g. activating T cell antigen,can result in a temporary interaction between the target cell and Tcell, thereby resulting in activation, e.g. cytotoxic activity, of the Tcell and subsequent lysis of the target cell.

In some embodiments, the T surface molecule, such as activating T cellantigen, is CD3 or is CD2. Specifically, a provided bispecificPD-1-binding polypeptide is capable of specifically binding anactivating T cell antigen expressed on a human T cell, such as human CD3or human CD3. In particular aspects, the additional binding domain thatis specific to the activating T cell antigen (e.g. CD3 or CD2) is anantibody or antigen-binding fragment. In some embodiments, aPD-1-binding polypeptide can be a bispecific antibody T cell-engagercontaining at least one PD-1 VHH domain that specifically binds to PD-1and an additional binding molecule that is an antibody orantigen-binding fragment specific for an activating component of a Tcell (e.g. a T cell surface molecule, e.g. CD3 or CD2).

Among bispecific antibody T cell-engagers are bispecific T cell engager(BiTE) molecules, which contain tandem scFv molecules fused by aflexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317,1255-1260 (2011); tandem scFv molecules fused to each other via, e.g. aflexible linker, and that further contain an Fc domain composed of afirst and a second subunit capable of stable association (WO2013026837);diabodies and derivatives thereof, including tandem diabodies (Holligeret al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293,41-66 (1999)); dual affinity retargeting (DART) molecules that caninclude the diabody format with a C-terminal disulfide bridge; ortriomabs that include whole hybrid mouse/rat IgG molecules (Seimetz etal, Cancer Treat Rev 36, 458-467 (2010). Similar formats of any of theabove molecules can be generated using any of the PD-1 VHH domainsprovided herein.

In some embodiments, the additional binding domain specific to anactivating T cell antigen is an antigen-binding fragment selected from aFab fragment, a F(ab′)₂ fragment, an Fv fragment, a scFv, disulfidestabilized Fv fragment (dsFv), a scAb, a dAb, a single domain heavychain antibody (VHH), or a single domain light chain antibody. In someembodiments, the additional binding domain is monovalent for binding theactivating T cell antigen, such as CD2 or CD3.

In some embodiments, the additional binding domain is capable of bindingto CD3 or a CD3 complex. A CD3 complex is a complex of at least fivemembrane-bound polypeptides in mature T-lymphocytes that arenon-covalently associated with one another and with the T-cell receptor.The CD3 complex includes the gamma, delta, epsilon, zeta, and eta chains(also referred to as subunits). In some embodiments, the additionalbinding molecule is an antibody or antigen-binding fragment capable ofspecifically binding to CD3 or a CD3 complex, also called a CD3-bindingdomain. In some embodiments, the CD3-binding domain capable of bindingCD3 or a CD3 complex includes one or more copies of an anti-CD3 Fabfragment, an anti-CD3 F(ab′)₂ fragment, an anti-CD3 Fv fragment, ananti-CD3 scFv, an anti-CD3 dsFv, an anti-CD3 scAb, an anti-CD3 dAb, ananti-CD3 single domain heavy chain antibody (VHH), and an anti-CD3single domain light chain antibody. In some embodiments, the anti-CD3binding domain is monovalent for binding CD3.

In some cases, the CD3-binding domain recognizes the CD3ε-chain. In someembodiments, the anti-CD3ε binding domain includes one or more copies ofan anti-CD3ε Fab fragment, an anti-CD3ε F(ab′)₂ fragment, an anti-CD3εFv fragment, an anti-CD3ε scFv, an anti-CD3ε dsFv, an anti-CD3ε scAb, ananti-CD3ε dAb, an anti-CD3ε single domain heavy chain antibody (VHH),and an anti-CD3ε single domain light chain antibody. In someembodiments, the anti-CD3ε binding domain is monovalent for bindingCD3ε.

Exemplary monoclonal antibodies against CD3 or a CD3 complex include,but are not limited to, OKT3, SP34, UCHT1 or 64.1, or an antigen-bindingfragment thereof (See e.g., June, et al., J. Immunol. 136:3945-3952(1986); Yang, et al., J. Immunol. 137:1097-1100 (1986); and Hayward, etal., Immunol. 64:87-92 (1988)). In some aspects, clustering of CD3 on Tcells, e.g., by immobilized or cell-localized or tetheredanti-CD3-antibodies, leads to T cell activation similar to theengagement of the T cell receptor but independent from its clone typicalspecificity. In one embodiment, the CD3-binding domain monovalently andspecifically binds a CD3 antigen, and is derived from OKT3(ORTHOCLONE-OKT3™ (muromonab-CD3); humanized OKT3 (U.S. Pat. No.7,635,475 and published international application No. WO2005040220);SP34 (Pessano et al. The EMBO Journal. 4: 337-344, 1985); humanizedvariant of SP34 (WO2015001085); Teplizumab™ (MGA031, Eli Lilly); ananti-CD3 binding molecule described in US2011/0275787; UCHT1 (Pollard etal. 1987 J Histochem Cytochem. 35(11):1329-38; WO2000041474); NI0401(WO2007/033230); visilizumab (U.S. Pat. No. 5,834,597); BC-3 (Anasettiet al., Transplantation 54: 844 (1992); H2C (described in PCTpublication no. WO2008/119567); V9 (described in Rodrigues et al., Int JCancer Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297)). Otheranti-CD3 antibodies also can be used in the constructs provided herein,including any described in International published PCT application Nos.WO199404679, WO2008119567, WO2015095392, WO2016204966, WO2019133761;published patent application Nos. US20170369563, US20180194842,US20180355038; U.S. Pat. Nos. 7,728,114, 7,381,803, 7,994,289.

In some embodiments, the CD3-binding domain contains a variable heavy(VH) chain set forth in SEQ ID NO: 19 and/or a variable light chain setforth in SEQ ID NO:20, or VH and/or VL sequences having at least 60%,70%, 80%, 90%, 95%, 97%, 98%, 99% identity with these sequences andspecifically binds CD3. In some embodiments, the CD3-binding domaincontains a CDRH1, CDRH2 and CDRH3 of the variable heavy (VH) chain setforth in SEQ ID NO:19 and a CDRL1, CDRL2 and CDRL3 variable light chainset forth in SEQ ID NO:20. In some cases, the CD3-binding regioncomprises a humanized version of the VH sequence set forth in SEQ IDNO:209 and a humanized version of the VL sequence set forth in SEQ IDNO:210. In some embodiments a CD3-binding region can contain a humanizedOKT3 derived VH domain sequence set forth in any one of SEQ ID NOs 21,22, 23 and/or a VL domain sequence set forth in any one of SEQ ID NOs24, 25, 26, or VH and/or VL sequences having at least 60%, 70%, 80%,90%, 95%, 97%, 98%, 99% identity with these sequences and specificallybinds CD3. In some embodiments, the CD3-binding domain is a Fab, scFv,Fv or dsFv, in which is contained any combination of the above VH and VLsequence, particularly any combination of a VH sequence set forth in anyof SEQ ID NOS: 21, 22, 23 and a VL sequence set forth in any of SEQ IDNOS: 24, 25, 26.

In some embodiments, the anti-CD3ε binding domain includes a VH CDR1sequence that includes at least the amino acid sequence TYAMN (SEQ IDNO: 29); a VH CD2 sequence that includes at least the amino acidsequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 30); a VH CDR3 sequence thatincludes at least the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:31), a VL CDR1 sequence that includes at least the amino acid sequenceRSSTGAVTTSNYAN (SEQ ID NO: 32); a VL CDR2 sequence that includes atleast the amino acid sequence GTNKRAP (SEQ ID NO: 33); and a VL CDR3sequence that includes at least the amino acid sequence ALWYSNLWV (SEQID NO: 34). In some embodiments, the CD3-binding domain is a Fab, scFv,Fv or dsFv, in which is contained a VH CDR1 sequence that includes atleast the amino acid sequence TYAMN (SEQ ID NO: 29); a VH CD2 sequencethat includes at least the amino acid sequence RIRSKYNNYATYYADSVKD (SEQID NO: 30); a VH CDR3 sequence that includes at least the amino acidsequence HGNFGNSYVSWFAY (SEQ ID NO: 31), a VL CDR1 sequence thatincludes at least the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO:32); a VL CDR2 sequence that includes at least the amino acid sequenceGTNKRAP (SEQ ID NO: 33); and a VL CDR3 sequence that includes at leastthe amino acid sequence ALWYSNLWV (SEQ ID NO: 34).

In some embodiments, the CD3-binding domain contains a variable heavy(VH) chain set forth in SEQ ID NO:27 and/or a variable light chain setforth in SEQ ID NO:28, or VH and/or VL sequences having at least 60%,70%, 80%, 90%, 95%, 97%, 98%, 99% identity with these sequences andspecifically binds to CD3. In some embodiments, the CD3-binding domaincontains a CDRH1, CDRH2 and CDRH3 of the variable heavy (VH) chain setforth in SEQ ID NO:27 and a CDRL1, CDRL2 and CDRL3 variable light chainset forth in SEQ ID NO:28. In some embodiments, the CD3-binding domaincontains a CDRH1, CDRH2 and CDRH3 set forth in SEQ ID NOs:29, 30 and 31,respectively and a CDRL1, CDRL2 and CDRL3 variable light chain set forthin SEQ ID NO:32, 33, and 34, respectively. In some cases, theCD3-binding region comprises a humanized version of the VH sequence setforth in SEQ ID NO:27 and a humanized version of the VL sequence setforth in SEQ ID NO:28. In some embodiments a CD3-binding region cancontain a humanized VH domain sequence set forth in any one of SEQ IDNOs 35-65 and/or a VL domain sequence set forth in any one of SEQ IDNOs: 66-84, or VH and/or VL sequences having at least 60%, 70%, 80%,90%, 95%, 97%, 98%, 99% identity with these sequences and specificallybinds to CD3. In some embodiments, the anti-CD3ε binding domain thereofincludes a variable heavy chain (Hv) comprising the amino acid sequenceof SEQ ID NO: 47 and a variable light chain (Lv) comprising the aminoacid sequence of SEQ ID NO: 75.

In some embodiments, the CD3-binding domain is a Fab, scFv, Fv or dsFv,in which is contained any combination of the above VH and VL sequence,particularly any combination of a VH sequence set forth in any of SEQ IDNOS: 35-65 and a VL sequence set forth in any of SEQ ID NOS: 66-84. Insome embodiments, the anti-CD3 binding domain is a Fab, scFv, Fv ordsFv, in which is contained a variable heavy chain (VH) comprising theamino acid sequence of SEQ ID NO: 47 and a variable light chain (VL)comprising the amino acid sequence of SEQ ID NO: 75.

In some embodiments, the CD3-binding domain contains a variable heavy(VH) chain set forth in any one of SEQ ID NO:313, 314, 317, or 318. Insome embodiments, the CD3-binding domain contains a variable light (VL)chain set forth in any one of SEQ ID NO:315, 316, 319, or 320.

The provided bispecific constructs can be formatted in any of a numberof formats containing the at least one PD-1 VHH domain and the at leastone additional domain specific to an activating T cell antigen, such asa CD3-binding domain.

In one embodiment, the bispecific construct is a bispecificsingle-domain antibody-linked Fab (S-Fab) containing at least one PD-1VHH domain as described linked, directly or indirectly to a Fab antigenbinding fragment specific to a T cell activating antigen, e.g. CD3, suchas an anti-CD3 Fab. The Fab against a T cell activating antigen, e.g.anti-CD3 Fab, can contain any of the VH and VL sequences as described.In some embodiments, the PD-1 VHH domain is linked to the C-terminus ofthe VH or VL chain of an anti-CD3 Fab. In some embodiments, the S-Fabcan be further modified, such as by conjugation with polyethylene glycol(PEG), N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers, proteins(such as albumin), polyglutamic acid or PASylation (Pan et al. (2018)International Journal of Nanomedicine, 2018:3189-3201).

In another embodiment, the bispecific construct is a scFv-single domainantibody in which the construct contains at least one PD-1 VHH asdescribed linked, directly or indirectly, to an scFv containing a VH anda VL of an antigen binding domain specific to a T cell activatingantigen, e.g. CD3. The scFv against a T cell activating antigen, e.g.anti-CD3 scFv, can contain any of the VH and VL sequences as described.In some embodiments, the VHH domain and the scFv are connected by alinker, such as a peptide linker. In some embodiments, the peptidelinker can be a peptide linker as described herein. In some embodiments,the VHH domain and the scFv are each connected, optionally through ahinge region or a linker (e.g. peptide linker), to an Fc region, such asan N-terminus of an Fc region. The Fc region can be any describedherein, such as a human Fc region or a variant thereof, e.g. a humanIgG1 Fc region or a variant thereof. In particular examples, the Fcregion is formed by variant Fc domains, e.g. variant human IgG1 domains,that are mutated or modified to promote heterodimerization in whichdifferent polypeptides can be dimerized to yield a heterodimer.

In a further embodiment, the CD3-binding domain is a single domainantibody, such as is a VHH domain that specifically binds to CD3. Singledomain antibodies, including VHH domains that bind to CD3 are known, seee.g. published U.S. patent application No. US20160280795. In someembodiments, the CD3-binding domain is an anti-CD3 VHH set forth in SEQID NO:85, or a sequence that exhibits at least 60%, 70%, 80%, 90%, 95%,97%, 98%, 99% identity with SEQ ID NO:85 and specifically binds to CD3.In such aspects, a bispecific construct provided herein can include atleast one PD-1 VHH domain and at least one CD3 VHH domain. Forformatting the constructs, in some cases, each VHH domain is connected,optionally through a hinge region or linker (e.g. peptide linker), to anFc region, such as an N-terminus of an Fc region. The Fc region can beany described herein, such as a human Fc region or a variant thereof,e.g. a human IgG1 Fc region or a variant thereof. In particularexamples, the Fc region is formed by variant Fc domains, e.g. varianthuman IgG1 domains, that are mutated or modified to promoteheterodimerization in which different polypeptides can be dimerized toyield a heterodimer.

In the above embodiments, exemplary modifications of an Fc region topromote heterodimerization are known, including any as described below,e.g. Table 3. In some embodiments, one Fc polypeptide of a heterodimericFc comprises the sequence of amino acids set forth in any of SEQ ID NOS:103, 107, 115 or 117, and the other Fc polypeptide of the heterodimericFc contains the sequence of amino acids set forth in any of SEQ IDNOS:104, 108, 111, 113, 119 or 121. In some embodiments, one Fcpolypeptide of a heterodimeric Fc comprises the sequence of amino acidsset forth in any of SEQ ID NOS: 105, 109, 116 or 118 and the other Fcpolypeptide of the heterodimeric Fc comprises the sequence of aminoacids set forth in any of SEQ ID NOS: 106, 110, 112, 114, 120 or 122.

2. Constrained CD3 Multispecific Construct

In some embodiments, the PD-1-binding polypeptide is a multispecificpolypeptide construct that is a constrained T-cell engaging fusionprotein. In particular aspects, the constrained multispecific constructsprovided herein bind an activating T cell antigen, such as a CD3, andPD-1. Typically, the provided constrained multispecific polypeptideconstructs also contain at least one antigen binding domain that binds atumor associated antigen (TAA). The constrained multispecificpolypeptide constructs provided herein include at least a firstcomponent that includes an immunoglobulin Fc region, a second componentthat includes one or more copies of at least a binding domain that bindsCD3 (referred to herein as an anti-CD3 binding domain or a CD3 bindingdomain, which are terms that are used interchangeably herein), and alinker, such as a polypeptide linker, that joins the first component andthe second component. In the provided multispecific polypeptideconstructs, one or both of the first and second components contain atleast one provided VHH domain that binds to PD-1, and one or both of thefirst and second components contain at least one TAA antigen bindingdomain, which, when engaged upon binding to antigen, render theconstrained CD3 binding region substantially able to bind CD3

In some embodiments, the constrained multispecific polypeptideconstructs provided herein exist in two states in terms of capacity tobind CD3 and subsequently activate T-cells: (1) the “inactive” stateoccurs when there is no binding of any or all of the antigen bindingdomain(s) to PD-1, such that the CD3 binding is constrained and T-cellinteraction is obviated or reduced, and (2) the “active” state occursupon antigen binding by any or all of the antigen binding domain(s),such that the CD3 binding region is able to bind CD3 and the T-cellinteraction is allowed.

In some embodiments, the Fc region is linked to the CD3 binding domainvia a linker or linkers. In some embodiments, the Fc region is linked tothe CD3 binding region via a non-cleavable linker or linkers. In someembodiments, the Fc region is linked to the CD3 binding region via acleavable linker or an otherwise labile linker or linkers. In someembodiments, cleavable linker is a linker that can be specificallycleaved in the presence of a protease. In some aspects, enhanced CD3binding occurs following cleavage of the cleavable linker. In some suchaspects, the “active” state can be further amplified via severalmechanisms, including via cleavage of the linker joining the CD3 bindingregion and the Fc region. In some embodiments, the cleavable linker is alinker that contains a substrate recognition site for a protease. Insome embodiments, wherein the Fc region and the CD3 binding region arelinked by a cleavable linker, enhanced CD3 binding may occur followingcleavage within the linker(s).

Further, in aspects wherein the Fc region and the CD3 binding region areoperably linked by a cleavable linker, cleavage of the linker(s) betweenthe Fc region and the CD3 binding region may separate the constrainedmultispecific polypeptide constructs into a first and second component.Depending on the composition of the constrained multispecificpolypeptide construct, the first and second component may have distinctfunctionalities. In some embodiments, the Fc region is a region thatexhibits one or more effector functions, such as ADCC, CDC or ADCPfunctions. In such examples, the constrained multispecific polypeptideconstructs of the disclosure can be used to produce a self-amplifyingsystem. For example, in some aspects, the incorporation of a proteasecleavable linker between the Fc and the components of the CD3 bindingdomain enables for amplification of the T-cell activating capacity byallowing full exposure of the CD3 binding domain. Depending on thespecific linker included, the amplification step can be mediated bytumor associated proteases or by granzymes released following antigendependent-T-cell activation. If a tumor protease cleavable linker isincluded the amplification is mediated by the tumor ortumor-microenvironment. Whereas, if a granzyme B cleavable linker isincluded the amplification may be self-mediated by T-cells followingantigen-dependent activation. Furthermore, in cases wherein an effectorenabled Fc is included in the construct, amplification may be mediatedby granzymes released from NK cell that occurs through an ADCCmechanism.

The provided constrained multispecific polypeptide constructs include aconfiguration in which the first component containing the Fc region isN-terminal to the second component containing the CD3 binding region. Insuch an embodiment, the first and second components are joined via alinker that is C-terminal to the end of the Fc region. In someembodiments, the at least one PD-1 VHH domain is positioned on theamino-terminal (N-term) region of the multispecific polypeptideconstruct. In some embodiments, the at least one PD-1 VHH domain ispositioned on the carboxy-terminal (C-term) region of the multispecificpolypeptide construct. In some embodiments, the constrainedmultispecific polypeptide construct contains only one PD-1 VHH domainsthat is positioned on either the N- or C-terminal regions of themultispecific polypeptide construct. n some embodiments, the at leastone TAA antigen binding domain is positioned on the amino-terminal(N-term) region of the multispecific polypeptide construct. In someembodiments, the at least one TAA antigen binding domain is positionedon the carboxy-terminal (C-term) region of the multispecific polypeptideconstruct. In some embodiments, the constrained multispecificpolypeptide construct contains at least two TAA antigen binding domainsthat are positioned on both the N- and C-terminal regions of themultispecific polypeptide construct.

In some embodiments, the constrained multispecific polypeptide constructis a dimer, in which dimerization is formed by covalent or non-covalentinteractions between two polypeptide chains. In some embodiments, thetwo polypeptide chains are covalently bonded to each other by, forexample, interchain disulfide bonds. In some embodiments, the Fc regionmediates dimerization via interchain disulfide bonds. In particularembodiments, a constrained multispecific polypeptide construct containsa heterodimeric Fc region in which, in some cases, the polypeptidechains of the multispecific polypeptide construct are different(heterodimer). In particular examples of a heterodimeric multispecificpolypeptide construct, the CD3-binding region is a two chain polypeptidecontaining a VH and a VL chain, such as is an Fv antibody fragmentcontaining the VH and VL. In some embodiments, the Fv antibody fragmentincludes a disulfide stabilized anti-CD3 binding Fv fragment (dsFv).

In some embodiments, a constrained multispecific polypeptide constructis formed from or includes two polypeptides, including a firstpolypeptide comprising a first Fc polypeptide of a heterodimeric Fcregion, a linker (e.g. cleavable or non-cleavable linker), a VH domainof an anti-CD3 antibody or antigen binding fragment (e.g. Fv); and asecond polypeptide comprising a second Fc polypeptide of theheterodimeric Fc region, the linker (e.g. the cleavable or non-cleavablelinker), a VL domain of the anti-CD3 antibody or antigen bindingfragment (e.g. Fv). In some embodiments, the first polypeptide containsone or two VHH domains that bind to PD-1. In some embodiments, thesecond polypeptide contains one or two VHH domains that bind to PD-1. Insome embodiments, a constrained multispecific polypeptide constructcontains only one PD-1 VHH domains. In some embodiments, the firstpolypeptide contains one or two TAA antigen binding domains. In someembodiments, the second polypeptide contains one or two TAA antigenbinding domains. In some embodiments, a constrained multispecificpolypeptide construct contains at least two TAA antigen binding domains.In some cases, at least one TAA antigen binding domain is locatedN-terminally to the Fc polypeptide and at least one TAA antigen bindingdomain is located C-terminally to the chain of the CD3-binding region.In particular embodiments, the at least one VHH domain that binds toPD-1 is on a separate polypeptide of the heterodimeric molecule than thepolypeptide containing the last least one, e.g. two, TAA antigen bindingdomains.

In some embodiments, a constrained multispecific polypeptide constructcontains at least two TAA antigen binding domains and at least one VHHdomain that binds PD-1. In some embodiments, a constrained multispecificpolypeptide construct contains (1) a first polypeptide comprising inorder of N-terminus to C-terminus: a first TAA antigen binding domain,the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. acleavable or non-cleavable linker), a chain (e.g. VH or VL) of ananti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv), and asecond TAA antigen binding domain; and (2) a second polypeptidecomprising in order of N-terminus to C-terminus: the second Fcpolypeptide of the heterodimeric Fc region, the same linker (e.g. samecleavable or non-cleavable linker), the other chain (other of the VH orVL) of the anti-CD3 antibody or antigen binding fragment, and a VHHdomain that binds PD-1. In some embodiments, a constrained multispecificpolypeptide construct contains (1) a first polypeptide comprising inorder of N-terminus to C-terminus: a first TAA antigen binding domain,the first Fc polypeptide of a heterodimeric Fc region, a linker (e.g. acleavable or non-cleavable linker), a chain (e.g. VH or VL) of ananti-CD3 antibody or antigen binding fragment (e.g. Fv or dsFv), and asecond TAA antigen binding domain; and (2) a second polypeptidecomprising in order of N-terminus to C-terminus: a VHH domain that bindsPD-1, the second Fc polypeptide of the heterodimeric Fc region, the samelinker (e.g. same cleavable or non-cleavable linker), and the otherchain (other of the VH or VL) of the anti-CD3 antibody or antigenbinding fragment.

In some embodiments, the first polypeptide or second polypeptide or boththe first and second polypeptide further include a co-stimulatoryreceptor binding region (CRBR) that binds a co-stimulatory receptor. Insome embodiments, the CRBR of the first and/or second polypeptide can belocated N-terminally to the Fc polypeptide and/or C-terminally to thechain of the CD3-binding region.

In some embodiments, a constrained multispecific polypeptide constructcontains at least two antigen binding domains that binds a TAA, aco-stimulatory receptor binding region (CRBR) that binds aco-stimulatory receptor and a VHH domain that binds PD-1. In someembodiments, a constrained multispecific polypeptide construct contains(1) a first polypeptide comprising in order of N-terminus to C-terminus:a first TAA antigen binding domain, the first Fc polypeptide of aheterodimeric Fc region, a linker (e.g. a cleavable linker), a chain(e.g. VH or VL) of an anti-CD3 antibody or antigen binding fragment(e.g. Fv or dsFv), and a second TAA antigen bind domain; and (2) asecond polypeptide comprising in order of N-terminus to C-terminus: oneof VHH domain that binds PD-1 or CRBR, the second Fc polypeptide of theheterodimeric Fc region, the same linker (e.g. same cleavable linker),the other chain (other of the VH or VL) of the anti-CD3 antibody orantigen binding fragment, and the other of the VHH domain that bindsPD-1 or CRBR.

Each of the components of the multispecific polypeptide constructs ofthe disclosure is described in more detail below.

a. Antigen Binding Domain

The multispecific polypeptide constructs of the present disclosureinclude at least one antigen binding domain, such as at least a firstantigen binding domain and a second antigen binding domain. In someaspects, the antigen binding domain, or independently each of theantigen binding domains, is selected from an antibody or antigen bindingfragment, a natural (or native) cognate binding partner, an Anticalin(engineered lipocalin), a Darpin, a Fynomer, a Centyrin (engineeredfibronectin III domain), a cystine-knot domain, an Affilin, an Affibody,or an engineered CH3 domain. In some embodiments, the natural cognatebinding partner comprises an extracellular domain or binding fragmentthereof of the native cognate binding partner of the TAA, or a variantthereof that exhibits binding activity to the TAA.

In some embodiments, the antigen binding domain, or independently eachof the antigen binding domains, such as the first antigen-binding domainand the second antigen binding domains, includes one or more copies ofan antibody or an antigen-binding fragment thereof. In some embodiments,the antigen binding domain or independently each of the antigen bindingdomains, such as the first antigen-binding domain and the second antigenbinding domains, includes one or more copies of an antibody or anantigen-binding fragment thereof selected from the group consisting of aFab fragment, a F(ab′)2 fragment, an Fv fragment, a scFv, a scAb, a dAb,a single domain heavy chain antibody, and a single domain light chainantibody.

In some embodiments, the antigen binding domain, or independently eachof the antigen binding domains, such as the first antigen-binding domainand the second antigen binding domains, is a single chain antibody. Insome examples, the single chain is an scFv, a scAb, a single domainheavy chain antibody, or a single domain light chain antibody.

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand the second antigen binding domains, includes one or more singledomain antibody (sdAb) fragments, for example VHH, VNAR, engineered VHor VK domains. VHHs can be generated from natural camelid heavy chainonly antibodies, genetically modified rodents that produce heavy chainonly antibodies, or naïve/synthetic camelid or humanized camelid singledomain antibody libraries. VNARs can be generated from cartilaginousfish heavy chain only antibodies. Various methods have been implementedto generate monomeric sdAbs from conventionally heterodimeric VH and VKdomains, including interface engineering and selection of specificgermline families.

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand/or the second antigen binding domains, of the multispecificpolypeptide constructs contains at least one sdAb or an scFv that bindsa TAA. In some embodiments, the at least one scFv or sdAb that binds aTAA is positioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct. In some embodiments, themultispecific polypeptide construct contains only one scFv or sdAb thatbinds to a TAA, which can be positioned either amino-terminally relativeto the Fc region and/or carboxy-terminally relative to the CD3 bindingregion. In some embodiments, the multispecific polypeptide constructcontains two scFvs or sdAbs that bind to a TAA, positionedamino-terminally relative to the Fc region and/or carboxy-terminallyrelative to the CD3 binding region. In some embodiments, themultispecific polypeptide construct contains three scFv or sdAb, inwhich two are positioned amino-terminally relative to the Fc region orcarboxy-terminally relative to the CD3 binding region, and the third ispositioned at the other end of the multispecific polypeptide construct.

In some embodiments, the multispecific polypeptide construct is formedfrom or includes two polypeptides, including a first polypeptidecomprising a first Fc polypeptide of a heterodimeric Fc region, alinker, a VH domain of an anti-CD3 antibody or antigen binding fragment(e.g. Fv), and an scFv or sdAb that binds to a tumor-associated antigen;and a second polypeptide comprising a second Fc polypeptide of theheterodimeric Fc region, the linker, a VL domain of the anti-CD3antibody or antigen binding fragment (e.g. Fv) and, optionally, the sameor different scFv or sdAb that binds to a tumor-associated antigen. ThescFv or sdAb that binds to a TAA can be positioned amino terminallyrelative to an Fc polypeptide of the heterodimeric Fc and/orcarboxy-terminally relative to a VH or VL chain of the CD3 bindingregion. At least one of the first and/or second polypeptide of themultispecific polypeptide construct also includes VHH domain that bindsPD-1, such as any set forth in the present disclosure. In some aspects,at least one of the first and/or second polypeptide of the multispecificpolypeptide construct may also include a CRBR that binds a costimulatoryreceptor or a chain thereof as described.

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand/or the second antigen binding domains, of the multispecificpolypeptide constructs contains binding domains as single domainantibodies (sdAbs).

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand the second antigen binding domains, contains more than one chain. Insome embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand/or the second antigen binding domains, of the multispecificpolypeptide constructs contains VH and VL sequences assembled as FABs.

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand/or the second antigen binding domains, of the multispecificpolypeptide constructs contains a VH-CH1 (Fd) and a VL-CL of a Fabantibody that binds a TAA. In some embodiments, the Fab antibodycontaining a VH-CH1 (Fd) and a VL-CL is positioned amino-terminallyrelative to the Fc region and/or carboxy-terminally relative to the CD3binding region of the multispecific polypeptide construct. In someembodiments, the multispecific polypeptide construct contains only oneFab antibody, containing a VH-CH1 (Fd) and VL-CL, that binds to a TAA,which can be positioned either amino-terminally relative to the Fcregion and/or carboxy-terminally relative to the CD3 binding region. Insome embodiments, the multispecific polypeptide construct contains twoFab antibody fragments, each containing a VH-CH1 (Fd) and VL-CL, thatbinds to a TAA, in which one is positioned amino-terminally relative tothe Fc region and the other is positioned carboxy-terminally relative tothe CD3 binding region.

In some embodiments, the multispecific polypeptide construct is formedfrom or includes three or more polypeptides, including a firstpolypeptide comprising a first Fc polypeptide of a heterodimeric Fcregion, a linker and a VH-CH1 (Fd) or VL-CL of a Fab antibody fragmentthat binds to a tumor-associated antigen; a second polypeptidecomprising a second Fc polypeptide of the heterodimeric Fc region, thelinker and, optionally, the same VH-CH1 (Fd) or VL-CL of the Fabantibody fragment that binds to a tumor-associated antigen, and a thirdpolypeptide comprising the other of the VH-CH1 (Fd) or VL-CL of the Fabantibody fragment that binds to the TAA.

In some embodiments, the antigen binding domain, or independently eachof the antigen binding domains, is or includes an extracellular domainor binding fragment thereof of the natural (or native) cognate bindingpartner of the TAA, or a variant thereof that exhibits binding activityto the TAA.

In some embodiments, the antigen binding domain or independently each ofthe antigen binding domains, such as the first antigen-binding domainand the second antigen binding domains, bind the same antigen. In someembodiments, there are more than one antigen binding domain that binds aTAA and each of the antigen binding domains, such as the firstantigen-binding domain and the second antigen binding domains, bind adifferent antigen. In some embodiments, each of the antigen bindingdomains, such as the first antigen-binding domain and the second antigenbinding domains, bind the same tumor associated antigen (TAA). In someembodiments, each of the antigen binding domains, such as the firstantigen-binding domain and the second antigen binding domains bind adifferent TAA. In some embodiments, the each of the antigen bindingdomains, such as the first antigen-binding domain and the second antigenbinding domains bind a different epitope on the same TAA. In someembodiments, each of the antigen binding domains, such as the firstantigen-binding domain and the second antigen binding domains, bind thesame epitope on the same TAA.

In some embodiments, the antigen binding domain, or independently eachof the antigen binding domains that binds TAA results in monovalent,bivalent, trivalent, or tetravalent binding to the TAA.

In some embodiments, the TAA is selected from the group consisting of1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor,APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9,(Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20,CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44,CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81,CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138,CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10,CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM,EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2,FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRα), GAL3ST1, G-CSF,G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors,Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM,Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor(FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R,IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R,Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin,MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, NotchReceptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta,TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha,TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor,TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) folate receptor alpha (FRα). For example, the antigenbinding domain contains the binding domain as an sdAb that binds FRα.Exemplary FRα-binding sdAbs are set forth in SEQ ID NOS: 86, 87, or 88.The antigen binding domain, or independently each antigen bindingdomain, in a provided multispecific polypeptide construct can have atleast 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to any the foregoing SEQ ID No and bindFRα.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) cMET. For example, the antigen binding domain contains thebinding domain as a sdAb that binds cMET. An exemplary cMET-binding sdAbis set forth in SEQ ID NO: 89 (U.S. Pat. No. 9,346,884). The antigenbinding domain, or independently each antigen binding domain, in aprovided multispecific polypeptide construct can have at least 85%, 85%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to a the foregoing SEQ ID No and bind cMET.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) B7H3. For example, the antigen binding domain contains thebinding domain as an scFv that binds B7H3. In some embodiments, theantigen binding domain is or contains a Fab antibody fragment comprisinga VH-CH1 (Fd) and LC. An exemplary B7H3 Fd is described in PCTPublication No, WO2017/030926.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) CD20. For example, the antigen binding domain contains thebinding domain as an scFv that binds CD20. Exemplary CD20-binding scFvsare set forth in SEQ ID NO: 90 or containing a VL and VH set forth inSEQ ID NO: 91 and 92 (U.S. Pub. No. US 2005/0123546). The antigenbinding domain, or independently each antigen binding domain, in aprovided multispecific polypeptide construct can have at least 85%, 85%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to any of the foregoing SEQ ID Nos and bind CD20.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) DLL3. For example, the antigen binding domain contains thebinding domain as an scFv that binds DLL3. Exemplary DLL3-binding scFvis set forth in SEQ ID NO: 93 and 94 (U.S. Pub. No. US 2017/0037130). Insome embodiments, the antigen binding domain is or contains a Fabantibody fragment comprising a Fd and LC that binds DLL3. An exemplaryDLL3 Fd is set forth in SEQ ID NO: 95 and an exemplary DLL3 LC is setforth in SEQ ID NO: 96 (U.S. Pat. No. 8,044,178). The antigen bindingdomain, or independently each antigen binding domain, in a providedmultispecific polypeptide construct can have at least 85%, 85%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to any of the foregoing SEQ ID Nos and bind DLL3.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) 5T4. An exemplary 5T4 Fd is set forth in SEQ ID NO: 97 andan exemplary 5T4 LC is set forth in SEQ ID NO: 98. In some embodiments,the antibody binding domain comprises a VH and VL as set forth in SEQ IDNOS: 99 and 100 (U.S. Pat. No. 8,044,178). The antigen binding domain,or independently each antigen binding domain, in a providedmultispecific polypeptide construct can have at least 85%, 85%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to any of the foregoing SEQ ID Nos and bind 5T4.

In some embodiments, at least one antigen binding domain, orindependently each antigen binding domain, binds the tumor associatedantigen (TAA) gpNMB. In some embodiments, the antigen binding domain isor contains a Fab fragment comprising a Fd and LC chain. An exemplarygpNMB Fd is set forth in SEQ ID NO: 101 and an exemplary gpNMB LC is setforth in SEQ ID NO: 102. The antigen binding domain, or independentlyeach antigen binding domain, in a provided multispecific polypeptideconstruct can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoingSEQ ID Nos and bind gpNMB.

In some embodiments, the antigen binding domain is linked, directly orindirectly via a linker, to the Fc region and/or to the CD3 bindingregion. In some embodiments, linkage is via a linker. In someembodiments, the linker is a linking peptide (LP), which can include anyflexible or rigid linker as described. In some embodiments, the linkeris selected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ IDNO: 1); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 2); GGSGGSGGSGGS, i.e.,(GGS)₄ (SEQ ID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 4).In some embodiments, the linker is a flexible linker comprising Glycineresidues, such as, by way of non-limiting example, GG, GGG, GGGG (SEQ IDNO: 5), GGGGG (SEQ ID NO: 6), and GGGGGG (SEQ ID NO: 7). In someembodiments, the linker includes a combination of a GS-linker and aGlycine linker.

b. Fc Region

A constrained multispecific polypeptide construct includes animmunoglobulin Fc region. Generally, the constrained multispecificpolypeptide construct is a dimer formed by polypeptides, each containingan Fc. The Fc polypeptide can be any as set forth above. In particularembodiments, the Fc region is formed by Fc domains that are mutated ormodified to promote heterodimerization in which different polypeptidescan be dimerized to yield a heterodimer. Thus, in some embodiments, thedimer is a heterodimer in which two polypeptide chains of themultispecific polypeptide construct are different.

Various methods are known for promoting heterodimerization ofcomplementary Fc polypeptides, see e.g. Ridgway et al, Protein Eng.9:617-621 (1996); Merchant et al, Nat. Biotechnol. 16(7): 677-81 (1998);Moore et al. (2011) MAbs, 3:546-57; Von Kreudenstein et al. MAbs, (2013)5:646-54; Gunasekaran et al. (2010) J. Biol. Chem., 285:19637-46;Leaver-Fay et al. (2016) Structure, 24:641-51; Ha et al. (2016)Frontiers in Immunology, 7:1; Davis et al. (2010) Protein Eng Des Sel,23:195-202; published international PCT Appl. No. WO 1998/050431, WO2009/089004, WO2011143545 WO 2014/067011, WO 2012/058768, WO2018027025;published U.S. patent Appl. No. US20140363426, US20150307628,US20180016354, US20150239991; and U.S. patent Nos. U.S. Pat. Nos.5,731,168, 7,183,076, 9,701,759, 9,605,084, and 9,650,446. Methods topromote heterodimerization of Fc chains include mutagenesis of the Fcregion, such as by including a set of “knob-into-hole” mutations orincluding mutations to effect electrostatic steering of the Fc to favorattractive interactions among different polypeptide chains. For example,in some embodiments, the Fc polypeptides of a heterodimer includes amutation to alter charge polarity across the Fc dimer interface suchthat coexpression of electrostatically matched Fc chains supportfavorable attractive interactions thereby promoting desired Fcheterodimer formation, whereas unfavorable repulsive charge interactionssuppress unwanted Fc homodimer formation (Guneskaran et al. (2010) JBC,285: 19637-19646). When co-expressed in a cell, association between thechains is possible but the chains do not substantially self-associatedue to charge repulsion. Other strategies for generating a heterodimericFc include mixing human IgG and IgA CH3 domain segments to create acomplementary CH3 heterodimer, which is referred to as a SEED Fc.

In some embodiments, to promote heterodimerization both polypeptides ofthe Fc heterodimer contain paired or complementary amino acidmodifications. Exemplary paired amino acid modification of polypeptidesof an Fc fusion are set forth in Table 3.

TABLE 3 Paired amino acids of Heterodimeric Fc First Fc polypeptideSecond Fc Polypeptide T366W T366S/L368W/Y407V T366W/S354CT366S/L368A/Y407V/Y349C S364H/F405A Y349T/Y349F T350V/L351Y/F405A/Y407VT350V/T366L/K392L/T394W K360D/D399M/Y407A E345R/Q347R/T366V/K409VK409D/K392D D399K/E356K K360E/K409W Q347R/D399V/F405T L360E/K409W/Y349CQ347R/399V/F405T/S354C K370E/K409W E357N/D399V/F405T

In some embodiments, modifications include introduction of aprotuberance (knob) into a first Fc polypeptide and a cavity (hole) intoa second Fc polypeptide such that the protuberance is positionable inthe cavity to promote complexing of the first and second Fc-containingpolypeptides. Amino acids targeted for replacement and/or modificationto create protuberances or cavities in a polypeptide are typicallyinterface amino acids that interact or contact with one or more aminoacids in the interface of a second polypeptide.

In some embodiments, a first Fc polypeptide that is modified to containprotuberance (hole) amino acids include replacement of a native ororiginal amino acid with an amino acid that has at least one side chainwhich projects from the interface of the first Fc polypeptide and istherefore positionable in a compensatory cavity (hole) in an adjacentinterface of a second polypeptide. Most often, the replacement aminoacid is one which has a larger side chain volume than the original aminoacid residue. One of skill in the art knows how to determine and/orassess the properties of amino acid residues to identify those that areideal replacement amino acids to create a protuberance. In someembodiments, the replacement residues for the formation of aprotuberance are naturally occurring amino acid residues and include,for example, arginine (R), phenylalanine (F), tyrosine (Y), ortryptophan (W). In some examples, the original residue identified forreplacement is an amino acid residue that has a small side chain suchas, for example, alanine, asparagine, aspartic acid, glycine, serine,threonine, or valine.

In some embodiments, a second Fc polypeptide that is modified to containa cavity (hole) is one that includes replacement of a native or originalamino acid with an amino acid that has at least one side chain that isrecessed from the interface of the second polypeptide and thus is ableto accommodate a corresponding protuberance from the interface of afirst polypeptide. Most often, the replacement amino acid is one whichhas a smaller side chain volume than the original amino acid residue.One of skill in the art knows how to determine and/or assess theproperties of amino acid residues to identify those that are idealreplacement residues for the formation of a cavity. Generally, thereplacement residues for the formation of a cavity are naturallyoccurring amino acids and include, for example, alanine (A), serine (S),threonine (T) and valine (V). In some examples, the original amino acididentified for replacement is an amino acid that has a large side chainsuch as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

The CH3 interface of human IgG1, for example, involves sixteen residueson each domain located on four anti-parallel β-strands which buries 1090Å2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry,20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgwayet al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168).Modifications of a CH3 domain to create protuberances or cavities aredescribed, for example, in U.S. Pat. No. 5,731,168; International PatentApplications WO98/50431 and WO 2005/063816; and Ridgway et al., (1996)Prot. Engin., 9: 617-621. In some examples, modifications of a CH3domain to create protuberances or cavities are typically targeted toresidues located on the two central anti-parallel β-strands. The aim isto minimize the risk that the protuberances which are created can beaccommodated by protruding into the surrounding solvent rather thanbeing accommodated by a compensatory cavity in the partner CH3 domain.

For example, in some embodiments the heterodimeric Fc includes apolypeptide having an amino acid modification within the CH3 domain atThr366, which when replaced with a more bulky amino acid, e.g., Try(T366W), is able to preferentially pair with a second CH3 domain havingamino acid modifications to less bulky amino acids at positions Thr366,Leu368, and Tyr407, e.g., Ser, Ala and Val, respectively(T366S/L368A/Y407V). Heterodimerization via CH3 modifications can befurther stabilized by the introduction of a disulfide bond, for exampleby changing Ser354 to Cys (S354C) and Tyr349 to Cys (Y349C) on oppositeCH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods,248: 7-15).

The resulting constrained multispecific polypeptide constructs can bepurified by any suitable method such as, for example, by affinitychromatography over Protein A or Protein G columns. Where two nucleicacid molecules encoding different polypeptides are transformed intocells, formation of homo- and heterodimers will occur. Conditions forexpression can be adjusted so that heterodimer formation is favored overhomodimer formation.

Techniques for recovery of heterodimers from homodimers based on adifferential affinity of the heterodimers for an affinity reagent areknown. In some aspects, such techniques include designing a heterodimerso that one of the Fc polypeptide chains does not bind to the affinityreagent protein A. In some cases, one of the polypeptide chain cancontain one or more amino acid substitution to abrogate or reduceaffinity for the protein A reagent in one of the polypeptides of the Fcheterodimer, see e.g. WO2017134440, WO2010151792, Jendeberg et al.(Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34. In some ofthese embodiments, the Fc region may be modified at the protein-Abinding site on one member of the heterodimer so as to prevent protein-Abinding and thereby enable more efficient purification of theheterodimeric fusion protein. An exemplary modification within thisbinding site is Ile253, for example Ile253Arg (I253R). In someembodiments, the modification may be H435R or H435R/Y436F. In someembodiments, an Fc polypeptide of an Fc heterodimer can contain amodification so that it is capable of binding protein A but not proteinG (pA+/pG−). Exemplary pA+/pG− amino acid modifications include an Fccontaining serine at position 428, serine at position 434 and optionallyhistidine at position 436, with reference to human IgGl or comprisingthese residues at the corresponding positions in human IgG 2, 3, or 4.In some aspects, such amino acid modifications in one IgG Fc polypeptideat positions 428, 434 and optionally 436 reduces or prevents the bindingof protein G, enhancing the purification of the protein.

In some embodiments, any of such modifications to confer differentialaffinity to an affinity reagent can be combined with any one or moreother amino acid modifications described above. For example, the I253Rmodification maybe combined with either the T366S/L368A/Y407Vmodifications or with the T366W modifications. The T366S/L368A/Y407Vmodified Fc is capable of forming homodimers as there is no stericocclusion of the dimerization interface as there is in the case of theT336W modified Fc. Therefore, in some embodiments, the I253Rmodification is combined with the T366S/L368A/Y407V modified Fc todisallow purification any homodimeric Fc that may have formed. Similarmodifications can be employed by combining T366S/L368A/Y407V and H453R.

In some embodiments, the Fc regions of the heterodimeric moleculeadditionally can contain one or more other Fc mutation, such as anydescribed above. In some embodiments, the heterodimer molecule containsan Fc region with a mutation that reduces effector function.

In some embodiments, one Fc polypeptide of a heterodimeric Fc comprisesthe sequence of amino acids set forth in any of SEQ ID NOS: 103, 107,115, or 117, and the other Fc polypeptide of the heterodimeric Fccontains the sequence of amino acids set forth in any of SEQ ID NOS:104, 108, 111, 113, 119 or 121. In some embodiments, one Fc polypeptideof a heterodimeric Fc comprises the sequence of amino acids set forth inany of SEQ ID NOS: 105, 109, 116 or 118 and the other Fc polypeptide ofthe heterodimeric Fc comprises the sequence of amino acids set forth inany of SEQ ID NOS: 106, 110, 112, 114, 120 or 122.

In some embodiments, the Fc region of the provided multispecificpolypeptide constructs exhibit one or more effector functions. In somecases, the Fc region is capable of providing Fc-mediated effectorfunctions, such as for example, ADCC (e.g., release of granzyme B by NKcells), ADCP, and/or CDC. In general, the Fc region is responsible foreffector functions, such as complement-dependent cytotoxicity (CDC) andantibody-dependent cell cytotoxicity (ADCC), in addition to theantigen-binding capacity, which is the main function of immunoglobulins.Additionally, the FcRn sequence present in the Fc region plays the roleof regulating the IgG level in serum by increasing the in vivo half-lifeby conjugation to an in vivo FcRn receptor. In some embodiments in whichthe multispecific polypeptide constructs contain a cleavable linker,cleavage of the linker can produce two components that each havebiological activity: the CD3-binding region that is able to bind andengage CD3 on a T cell, which, in some aspects, also can contain a CRBRfor inducing a costimulatory signal on the T cell and/or a VHH domainthat binds PD-1 for blocking an inhibitory signal on the T cell; and theFc region linked to the TAA antigen binding domain that can exhibittarget-specific effector function.

In some embodiments, the Fc region includes an Fc polypeptide that ismutated or modified to alter one or more effector functions. Thus, insome cases, effector functions such as on or more of ADCC, ADCP and/orCDC can be altered, such as reduced or enhanced, in an Fc for use withthe provided constrained multispecific polypeptide constructs. Exemplarymutations to reduce effector function include any as described above.

c. CD3 Binding Domain

A constrained multispecific polypeptide construct includes a CD3 bindingdomain. The anti-CD3 binding domains of the disclosure activate T cellsvia engagement of CD3 or a member of the CD3 complex on the T cells. Inpreferred embodiments, the anti-CD3 binding domains of the disclosurespecifically bind the epsilon chain of CD3, also known as CD3ε. Theanti-CD3ε binding domains of the disclosure activate T cells viaengagement of CD3ε on the T cells. The anti-CD3 binding domains of thedisclosure agonize, stimulate, activate, and/or otherwise augmentCD3-mediated T cell activation. Biological activities of CD3 include,for example, T cell activation and other signaling through interactionbetween CD3 and the antigen-binding subunits of the T-Cell Receptor(TCR). For example, the anti-CD3 binding domains of the disclosurecompletely or partially activate T cells via engagement of CD3ε on Tcells by partially or completely modulating, e.g., agonizing,stimulating, activating or otherwise augmenting CD3-mediated T cellactivation.

The CD3 binding domain can be any as described above. In particularembodiments, the CD3 binding domain is an Fv antibody fragment thatbinds CD3ε (referred to herein as an anti-CD3ε Fv fragment). In someembodiments, the anti-CD3ε Fv antibody fragment is a disulfidestabilized anti-CD3 binding Fv fragment (dsFv). In some embodiments, theanti-CD3 binding domain is monovalent for binding CD3.

In some embodiments, the CD3 binding region is an Fv antibody fragmentcontaining a variable heavy chain (Hv, also called VH) and variablelight chain (Lv, also called VL), such as any as described. In aspectsof such embodiments, the immunoglobulin Fc region is a heterodimeric Fcregion containing two different Fc polypeptides capable of heterodimericassociation between both polypeptides of the Fc heterodimer, such as anyas described. In such embodiments, the variable heavy chain (VH) andvariable light chain (VL) of the CD3 binding region are linked onopposite chains of the heterodimeric Fc.

In some embodiments, the CD3 binding region is an Fv or dsFv of SP34(Pessano et ai. The EMBO Journal. 4: 337-344, 1985) or of a humanizedvariant of SP34 (WO2015001085).

In some embodiments, the anti-CD3ε binding domain thereof is an Fv ordsFv fragment that includes a combination of heavy chain variable aminoacid sequence and a light chain variable amino acid sequence. In someembodiments, the CD3-binding domain is an Fv or dsFv fragment in whichis contained a VH CDR1 sequence that includes at least the amino acidsequence TYAMN (SEQ ID NO: 29); a VH CD2 sequence that includes at leastthe amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 30); a VH CDR3sequence that includes at least the amino acid sequence HGNFGNSYVSWFAY(SEQ ID NO: 31), a VL CDR1 sequence that includes at least the aminoacid sequence RSSTGAVTTSNYAN (SEQ ID NO: 32); a VL CDR2 sequence thatincludes at least the amino acid sequence GTNKRAP (SEQ ID NO: 33); and aVL CDR3 sequence that includes at least the amino acid sequenceALWYSNLWV (SEQ ID NO: 34). In some embodiments, the anti-CD3ε bindingdomain thereof is an Fv or dsFv fragment that includes a heavy chainvariable amino acid sequence selected from the group of SEQ ID NO: 35-65and a light chain variable amino acid sequence selected from the groupconsisting of SEQ ID NO: 66-84, 285. In some embodiments, the anti-CD3εbinding domain thereof is an Fv or dsFv fragment that includes a heavychain variable amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 35-65 and an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 66-84, 285 an amino acid sequence. In someembodiments, the anti-CD3 binding domain is an Fv or dsFv, in which iscontained a variable heavy chain (VH) comprising the amino acid sequenceof SEQ ID NO: 47 and a variable light chain (VL) comprising the aminoacid sequence of SEQ ID NO: 285.

d. Linker

A constrained multispecific polypeptide constructs contain a linker thatjoins or couples the first component containing the immunoglobulin Fcregion and the second component containing the CD3 binding region. Insome embodiments, the linker is positioned at the end of the C-terminalregion of the Fc region, such that the Fc region is N-terminal to theCD3 binding region. It is understood that because the providedconstrained multispecific polypeptide constructs are multimers, such asdimers containing a first and second polypeptide that together form thefirst and second component, the provided constructs include a linkerjoining the Fc portion and the CD3 binding region of the first and alinker joining the Fc portion and the CD3 binding region of the secondpolypeptide. In some embodiments, the first polypeptide includes a firstFc polypeptide of a heterodimeric Fc region, a linker, and a firstdomain (e.g. VH) of a CD3 binding region, and the second polypeptideincludes a second Fc polypeptide of the heterodimeric Fc region, alinker and second domain (e.g. VL) of the CD3 binding region. Typically,the linkers present in the first and second polypeptides of theconstrained multispecific polypeptide construct are the same. Thus, insome embodiments, each domain of the CD3 binding domain is linked via alinker, such as the same linker, to opposite polypeptides of the Fc,such as heterodimeric Fc.

Various polypeptide linkers for use in fusion proteins are known (seee.g. Chen et al. (2013) Adv. Drug. Deliv. 65:1357-1369; andInternational PCT publication No. WO 2014/099997, WO2000/24884; U.S.Pat. Nos. 5,258,498; 5,525,491; 5,525,491, 6,132,992).

In some embodiments, the linker is chosen so that, when the CD3 bindingregion is joined to the Fc region of the multispecific polypeptideconjugate, the CD3 binding region is constrained and not able to, or notsubstantially able to, bind or engage CD3 on the surface of a cell, e.g.T cell, upon contact of the multispecific polypeptide construct with thecell. Various assays can be employed to assess binding or engagement ofCD3 by the multispecific polypeptide construct, including assays toassess T cell binding, NFAT activation using a reporter system,cytolytic T cell activity, cytokine production and/or expression of Tcell activation markers. Exemplary assays are shown in the providedExamples. Typically, the linker also is one that ensures correct foldingof the polypeptide construct, does not exhibit a charge that would beinconsistent with the activity or function of the linked polypeptides orform bonds or other interactions with amino acid residues in one or moreof the domains that would impede or alter activity of the linkedpolypeptides. In some embodiments, the linker is a polypeptide linker.The polypeptide linker can be a flexible linker or a rigid linker or acombination of both. In some aspects, the linker is a short, medium orlong linker. In some embodiments, the linker is up to 40 amino acids inlength. In some embodiments, the linker is up to 25 amino acids inlength. In some embodiments, the linker is at least or is at least about2 amino acids in length. In some aspects, a suitable length is, e.g., alength of at least one and typically fewer than about 40 amino acidresidues, such as 2-25 amino acid residues, 5-20 amino acid residues,5-15 amino acid residues, 8-12 amino acid. In some embodiments, thelinker is from or from about 2 to 24 amino acids, 2 to 20 amino acids, 2to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 24 aminoacids, 6 to 20 amino acids, 6 to 18 amino acids, 6 to 14 amino acids, 6to 12 amino acids, 6 to 10 amino acids, 6 to 8 amino acids, 8 to 24amino acids, 8 to 20 amino acids, 8 to 18 amino acids, 8 to 14 aminoacids, 8 to 12 amino acids, 8 to 10 amino acids, 10 to 24 amino acids,10 to 20 amino acids, 10 to 18 amino acids, 10 to 14 amino acids, 10 to12 amino acids, 12 to 24 amino acids, 12 to 20 amino acids, 12 to 18amino acids, 12 to 14 amino acids, 14 to 24 amino acids, 14 to 20 aminoacids, 14 to 18 amino acids, 18 to 24 amino acids, 18 to 20 amino acidsor 20 to 24 amino acids. In some embodiments, the linker is 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids inlength.

In certain aspects, the longer the linker length, the greater the CD3binding when the multispecific polypeptide conjugate is bounds to itsantigen, e.g. TAA. Thus, in some aspects, the linker is greater than 12amino acids in length, such as greater than 13, 14, 15, 16, 17 or 18amino acids in length. In some embodiments, the linker is 12 to 40 aminoacids in length, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18acids, 12 to 15 amino acids, 15 to 40 amino acids, 15 to 30 amino acids,15 to 24 amino acids, 15 to 18 amino acids, 18 to 40 amino acids, 18 to30 amino acids, 18 to 24 amino acids, 24 to 40 amino acids, 24 to 30amino acids or 30 to 40 amino acids.

The linkers can be naturally-occurring, synthetic or a combination ofboth. Particularly suitable linker polypeptides predominantly includeamino acid residues selected from Glycine (Gly), Serine (Ser), Alanine(Ala), and Threonine (Thr). For example, the linker may contain at least75% (calculated on the basis of the total number of residues present inthe peptide linker), such as at least 80%, at least 85%, or at least 90%of amino acid residues selected from Gly, Ser, Ala, and Thr. The linkermay also consist of Gly, Ser, Ala and/or Thr residues only. In someembodiments, the linker contains 1-25 glycine residues, 5-20 glycineresidues, 5-15 glycine residues, or 8-12 glycine residues. In someaspects, suitable peptide linkers typically contain at least 50% glycineresidues, such as at least 75% glycine residues. In some embodiments, apeptide linker comprises glycine residues only. In some embodiments, apeptide linker comprises glycine and serine residues only.

In some embodiments, these linkers are composed predominately of theamino acids Glycine and Serine, denoted as GS-linkers herein. In someembodiments, the linker contains (GGS)n, wherein n is 1 to 10, such as 1to 5, for example 1 to 3, such as GGS(GGS)n (SEQ ID NO:123), wherein nis 0 to 10. In particular embodiments, the linker contains the sequence(GGGGS)n (SEQ ID NO: 123), wherein n is 1 to 10 or n is 1 to 5, such as1 to 3. In further embodiments, the linker contains (GGGGGS)n (SEQ IDNO:124), wherein n is 1 to 4, such as 1 to 3. The linker can includecombinations of any of the above, such as repeats of 2, 3, 4, or 5 GS,GGS, GGGGS, and/or GGGGGS linkers may be combined. In some embodiments,such a linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18 or 19 amino acids in length.

In some embodiments, the linker is (in one-letter amino acid code): GGS,GGGGS (SEQ ID NO: 125), or GGGGGS (SEQ ID NO: 126). In some embodiments,the GS-linker comprises an amino acid sequence of GGSGGS, i.e., (GGS)₂(SEQ ID NO: 1); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 2); GGSGGSGGSGGS,i.e., (GGS)₄ (SEQ ID NO: 3); GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO:4); GGGGGSGGGGGSGGGGGS, i.e., (G5S)₃ (SEQ ID NO: 127),GGSGGGGSGGGGSGGGGS (SEQ ID NO: 129) and GGGGSGGGGSGGGGS (SEQ ID NO:128).In some embodiments, the linker is GGGG (SEQ ID NO:5). In some of any ofthe above examples, serine can be replaced with alanine (e.g., (Gly4Ala)or (Gly3Ala)).

In some embodiments, the linker includes a peptide linker having theamino acid sequence Gly_(x)Xaa-Gly_(y)-Xaa-Gly_(z) (SEQ ID NO:130),wherein each Xaa is independently selected from Alanine (Ala), Valine(Val), Leucine (Leu), Isoleucine (Ile), Methionine (Met), Phenylalanine(Phe), Tryptophan (Trp), Proline (Pro), Glycine (Gly), Serine (Ser),Threonine (Thr), Cysteine (Cys), Tyrosine (Tyr), Asparagine (Asn),Glutamine (Gln), Lysine (Lys), Arginine (Arg), Histidine (His),Aspartate (Asp), and Glutamate (Glu), and wherein x, y, and z are eachintegers in the range from 1-5. In some embodiments, each Xaa isindependently selected from the group consisting of Ser, Ala, and Thr.In a specific variation, each of x, y, and z is equal to 3 (therebyyielding a peptide linker having the amino acid sequenceGly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly (SEQ ID NO:131), whereineach Xaa is selected as above.

In some embodiments, the linker is serine-rich linkers based on therepetition of a (SSSSG)y (SEQ ID NO:132) motif where y is at least 1,though y can be 2, 3, 4, 5, 6, 7, 8 and 9.

In some cases, it may be desirable to provide some rigidity into thepeptide linker. This may be accomplished by including proline residuesin the amino acid sequence of the peptide linker. Thus, in someembodiments, a linker comprises at least one proline residue in theamino acid sequence of the peptide linker. For example, a peptide linkercan have an amino acid sequence wherein at least 25% (e.g., at least 50%or at least 75%) of the amino acid residues are proline residues. In oneparticular embodiment, the peptide linker comprises proline residuesonly.

In some aspects, a peptide linker comprises at least one cysteineresidue, such as one cysteine residue. For example, in some embodiments,a linker comprises at least one cysteine residue and amino acid residuesselected from the group consisting of Gly, Ser, Ala, and Thr. In somesuch embodiments, a linker comprises glycine residues and cysteineresidues, such as glycine residues and cysteine residues only.Typically, only one cysteine residue will be included per peptidelinker. One example of a specific linker comprising a cysteine residueincludes a peptide linker having the amino acid sequenceGly_(m)-Cys-Gly_(n), wherein n and m are each integers from 1-12, e.g.,from 3-9, from 4-8, or from 4-7. In a specific variation, such a peptidelinker has the amino acid sequence GGGGG-C-GGGGG (SEQ ID NO: 133).

In some embodiments, the linker of the fusion protein is a structured orconstrained linker. In particular embodiments, the structured linkercontains the sequence (AP)n or (EAAAK)n (SEQ ID NO:134), wherein n is 2to 20, preferably 4 to 10, including but not limited to, AS-(AP)n-GT(SEQ ID NO:135) or AS-(EAAAK)n-GT (SEQ ID NO:136), wherein n is 2 to 20,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In otherembodiments, the linker comprises the sequences (GGGGA)n (SEQ IDNO:137), (PGGGS)n (SEQ ID NO:138), (AGGGS)n (SEQ ID NO:139) orGGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO:140, wherein n is 2 to 20. In someembodiments, the linker is SSSASASSA (SEQ ID NO:141), GSPGSPG (SEQ IDNO:142), or ATTTGSSPGPT (SEQ ID NO:143). In some embodiments, suchlinkers, by virtue of their structure, may be more resistant toproteolytic degradation, thereby offering an advantage when injected invivo.

In some embodiments, the linker is not a cleavable linker, also callednon-cleavable linker. In some embodiments, the linker is not a cleavableby a protease. In some embodiments, a linker that is not a cleavablelinker or that is not cleavable by a protease is one that is generallystable for in vivo delivery or recombinant production. In some aspects,a linker that is not cleavable by a protease includes those that do notcontain at least one peptide bond which preferably lies within acleavable peptide sequence or recognition site of a protease. Inparticular embodiments, a non-cleavable linker is not a target substratefor a protease, such that it is not preferentially or specificallycleaved by a protease compared to a linker that contains a substraterecognition site for the same protease.

In some embodiments, the linker does not contains a substraterecognition site or cleavage site for a particular protease, which isthe sequence recognized by the active site of a protease that is cleavedby a protease. Typically, for example, for a serine protease, a cleavagesequence is made up of the P1-P4 and P1′-P4′ amino acids in a substrate,where cleavage occurs after the P1 position. Typically, a cleavagesequence for a serine protease is six residues in length to match theextended substrate specificity of many proteases, but can be longer orshorter depending upon the protease. Typically, the linker does notinclude a P1-P1′ scissile bond sequence that is recognized by aprotease. In some aspects, a non-cleavable linker or a linker that doesnot contain a substrate recognition site that is specifically recognizedfor cleavage by a protease is one whose cleavage by a protease issubstantially less than cleavage of a target substrate of the protease.

In some embodiments, the linker is a cleavable linker. In some aspects,a cleavable linker is a linker, such as any described above, thatfurther includes a sequence that is a substrate for a protease due tothe presence of at least one bond that can be broken under physiologicalconditions. In some cases, a cleavable linker is susceptible to orsensitive to cleavage under specific conditions that exist in vivo, suchas following exposure to an extracellular protease, including thosepresent in cellular environments in vivo. In some cases, the proteasemay be present in a particular physiological microenvironment, such asthe tumor microenvironment, thereby restricting the sites at whichcleavage may occur.

A protease typically exhibits specificity or preference for cleavage ofa particular target substrate compared to another non-target substrate.Such a degree of specificity can be determined based on the rateconstant of cleavage of a sequence, e.g. linker, which is a measure ofpreference of a protease for its substrate and the efficiency of theenzyme. Any method to determine the rate of increase of cleavage overtime in the presence of various concentrations of substrate can be usedto calculate the specificity constant. For example, a substrate islinked to a fluorogenic moiety, which is released upon cleavage by aprotease. By determining the rate of cleavage at different proteaseconcentrations the specificity constant for cleavage (k_(cat)/K_(m)) canbe determined for a particular protease towards a particular linker. Insome embodiments, a cleavable linker is a linker that is capable ofbeing specifically cleaved by a protease at a rate of about at least1×10⁴ M⁻¹S⁻¹, or at least 5×10⁴ M⁻¹S, at least 10×10⁴ M⁻¹S. at least10×10⁵ M⁻¹S or more.

In some embodiments, a constrained multispecific polypeptide constructsof the disclosure include a cleavable linker that joins the first andsecond components. In some embodiments, the cleavable linker includes anamino acid sequence that can serve as a substrate for a protease,usually an extracellular protease. For example, the cleavable linker mayinclude a cleavage sequence containing at least one peptide bond whichpreferably lies within a cleavable peptide sequence of a protease.Suitable proteases include, for example, matrix metalloproteases (MMP),cysteine proteases, serine proteases and plasmin activators, which areformed or activated in intensified manner in diseases such as rheumatoidarthritis or cancer, leading to excessive tissue degradation,inflammations and metastasis. In particular embodiments, the protease isa protease that is produced by a tumor, an activated immune effectorcell (e.g. a T cell or a NK cell), or a cell in a tumormicroenvironment. In some embodiments, the protease is a granzyme B, amatriptase or an MMP, such as MMP-2.

The cleavable linker may be selected based on a protease that isproduced by a tumor that is in proximity to cells that express thetarget and/or produced by a tumor that is co-localized in tissue withthe desired target of the multispecific polypeptide constructs. Thereare reports in the literature of increased levels of proteases havingknown substrates in a number of cancers, e.g., solid tumors. See, e.g.,La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421.

In some embodiments, the cleavable linker that joins the first andsecond component of a constrained multispecific polypeptide construct iscleaved by a protease produced by an immune effector cell that isactivated by one of the components. For example, multispecificpolypeptide constructs that encompass an effector enabled or enhancedIgG Fc region are capable of eliciting ADCC when engaged with the targetantigen. Central to ADCC is the release of granzyme B and perforin fromthe effector cells, namely NK cells and cytotoxic T-cells. Upon releasegranzyme B enters the target cell in a perforin dependent manner whereinit mediates apoptosis. Importantly, granzyme B is active within theextracellular synapse between the effector cell and the target cell. Insome embodiments, the cleavable linker that joins the first and secondcomponent multispecific polypeptide construct is cleaved by granzyme B.Granzyme B is released during effector cell activation mediated by oneof the components of the multispecific polypeptide construct. In someembodiments, granzyme B and other proteases can be produced by immuneeffector cells, including activated T cells or NK cells. In someembodiments, activation of T cells by CD3 engagement upon binding of aTAA by a multispecific polypeptide construct may release such proteases,which then can cleave a specific cleavable linker thereby potentiatingor increasing activity of the CD3 binding molecule to engage CD3. Insome embodiments, the cleavage can amplify or increase the activityachieved by the multispecific construct when bound to TAA in anuncleaved state.

Exemplary substrates include but are not limited to substrates cleavableby one or more of the following enzymes or proteases: ADAMS, ADAMTS,e.g. ADAM8; ADAM9; ADAM10; ADAM12; ADAM15; ADAM17/TACE; ADAMDEC1;ADAMTS1; ADAMTS4; ADAMTS5; aspartate proteases, e.g., BACE or Renin;aspartic cathepsins, e.g., Cathepsin D or Cathepsin E; Caspases, e.g.,Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6,Caspase 7, Caspase 8, Caspase 9, Caspase 10, or Caspase 14; cysteinecathepsins, e.g., Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L,Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P; Cysteine proteinases,e.g., Cruzipain; Legumain; Otubain-2; KLKs, e.g., KLK4, KLK5, KLK6,KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; Metallo proteinases, e.g.,Meprin; Neprilysin; PSMA; BMP-1; MMPs, e.g., MMP1, MMP2, MMP3, MMP7,MMP8, MMP9, MMP10, MMP11, MMP12, MMPP13, MMP14, MMPP15, MMP16, MMPP17,MMPP19, MMP20, MMP23, MMP24, MMP26, or MMP27, serine proteases, e.g.,activated protein C, Cathepsin A, Cathepsin G, Chymase, coagulationfactor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), Elastase,granzyme B, Guanidinobenzoatase, HtrA1, Human Neutrophil Elastase,Lactoferrin, Marapsin, NS3/4A, PACE4, Plasmin, PSA, tPA, Thrombin,Tryptase, uPA; Type II Transmembrane Serine Proteases (TTSPs), e.g.,DESC1, DPP-4, FAP, Hepsin, Matriptase-2, Matriptase, TMPRSS2, TMPRSS3,or TMPRSS4; and any combination thereof.

In some embodiments, the cleavable linker is cleaved by multipleproteases, e.g., 2 or more proteases, 3 or more proteases, 4 or moreproteases, and so on.

In some embodiments, the cleavable linker is selected for use with aspecific protease, for example a protease that is known to be producedby a tumor that is in proximity to cells that express the target and/orproduced by a tumor that is co-localized with the target of themultispecific polypeptide construct.

In some embodiments, the cleavable linker contains a substraterecognition site or cleavage site for a particular protease, which isthe sequence recognized by the active site of a protease that is cleavedby a protease. Typically, for example, for a serine protease, a cleavagesequence is made up of the P1-P4 and P1′-P4′ amino acids in a substrate,where cleavage occurs after the P1 position. Typically, a cleavagesequence for a serine protease is six residues in length to match theextended substrate specificity of many proteases, but can be longer orshorter depending upon the protease. Typically, the cleavable linkerincludes a P1-P1′ scissile bond sequence that is recognized by aprotease. In some aspects, the cleavable linker is engineered tointroduce a peptide bond able to be cleaved by a specific protease, forexample by introducing a substrate recognition site sequence or cleavagesequence of the protease.

In some embodiments, the cleavable linker includes a combination of twoor more substrate sequences. In some embodiments, each substratesequence is cleaved by the same protease. In some embodiments, at leasttwo of the substrate sequences are cleaved by different proteases. Insome embodiments, the cleavable linker comprises an amino acid that is asubstrate for granzyme B. In some embodiments, a granzyme B cleavablelinker contains an amino acid sequence having the general formula P4 P3P2 P1↓P1′ (SEQ ID NO: 144), wherein P4 is amino acid I, L, Y, M, F, V,or A; P3 is amino acid A, G, S, V, E, D, Q, N, or Y; P2 is amino acid H,P, A, V, G, S, or T; P1 is amino acid D or E; and P1′ is amino acid I,L, Y, M, F, V, T, S, G or A. In some embodiments, a granzyme B cleavablelinker contains an amino acid sequence having the general formula P4 P3P2 P1↓P1′ (SEQ ID NO: 145), wherein P4 is amino acid I or L; P3 is aminoacid E; P2 is amino acid P or A; P1 is amino acid D; and P1′ is aminoacid I, V, T, S, or G.

In some embodiments, the substrate for granzyme B comprises the aminoacid sequence LEAD (SEQ ID NO: 146), LEPG (SEQ ID NO: 147), or LEAE (SEQID NO:148). In some embodiments, the cleavable linker contains the aminoacid sequence the cleavable linker comprises the amino acid sequenceIEPDI (SEQ ID NO: 149), LEPDG (SEQ ID NO:150), LEADT (SEQ ID NO:151),IEPDG (SEQ ID NO:152), IEPDV (SEQ ID NO:153), IEPDS (SEQ ID NO:154),IEPDT (SEQ ID NO:155), IEPDP (SEQ ID NO:144), LEPDG (SEQ ID NO:152) orLEADG (SEQ ID NO:153).

In some embodiments, the cleavable linker comprises an amino acid thatis a substrate for matriptase. In some embodiments, the cleavable linkercomprises the sequence P4QAR↓(A/V) (SEQ ID NO: 156), wherein P4 is anyamino acid. In some embodiments, the cleavable linker comprises thesequence RQAR(A/V) (SEQ ID NO: 157). In some embodiments, the substratefor matriptase comprises the amino acid sequence RQAR (SEQ ID NO: 158).In some embodiments, the cleavable linker comprises the amino acidsequence RQARV (SEQ ID NO: 159).

In some embodiments, the cleavable linker comprises an amino acid thatis a substrate for one or more matrix metalloproteases (MMPs). In someembodiments, the MMP is MMP-2. In some embodiments, the cleavable linkercontains. the general formula P3 P2 P1↓P1′ (SEQ ID NO: 160), wherein P3is P, V or A; P2 is Q or D; P1 is A or N; and P1′ is L, I or M. In someembodiments, the cleavable linker contains the general formula P3 P2P1↓P1′ (SEQ ID NO: 161), wherein P3 is P; P2 is Q or D; P1 is A or N;and P1′ is L or I. In some embodiments, the substrate for MMP comprisesthe amino acid sequence PAGL (SEQ ID NO: 162).

In some embodiments, the cleavable linker comprises a combination of anamino acid sequence that is a substrate for granzyme B and an amino acidsequence that is a substrate for matriptase. In some embodiments, thecleavable linker comprises a combination of the amino acid sequence LEAD(SEQ ID NO: 146) and the amino acid sequence RQAR (SEQ ID NO: 158).

In some embodiments, the cleavable linker comprises a combination of anamino acid sequence that is a substrate for granzyme B and an amino acidsequence that is a substrate for MMP. In some embodiments, the cleavablelinker comprises a combination of the amino acid sequence LEAD (SEQ IDNO: 146) and the amino acid sequence PAGL (SEQ ID NO: 162).

In some embodiments, the cleavable linker comprises a combination of anamino acid sequence that is a substrate for matriptase and an amino acidsequence that is a substrate for MMP. In some embodiments, the cleavablelinker comprises a combination of the amino acid sequence RQAR (SEQ IDNO: 158) and the amino acid sequence PAGL (SEQ ID NO: 162).

In some embodiments, the cleavable linker comprises a combination of anamino acid sequence that is a substrate for granzyme B, an amino acidsequence that is a substrate for matriptase, and an amino acid sequencethat is a substrate for MMP. In some embodiments, the cleavable linkercomprises a combination of an amino acid sequence that is a substratefor granzyme B and an amino acid sequence that is a substrate for MMP.In some embodiments, the cleavable linker comprises a combination of theamino acid sequence LEAD (SEQ ID NO: 146), the amino acid sequence RQAR(SEQ ID NO: 158), and the amino acid sequence PAGL (SEQ ID NO: 162).

The cleavable linker can include any known linkers. Examples ofcleavable linkers are described in Be'liveau et al. (2009) FEBS Journal,276; U.S. published application Nos. US20160194399; US20150079088;US20170204139; US20160289324; US20160122425; US20150087810;US20170081397; U.S. Pat. No. 9,644,016.

In some embodiments, the cleavable linker comprises an amino acidsequence selected from the group consisting of TGLEADGSPAGLGRQARVG (SEQID NO: 163); TGLEADGSRQARVGPAGLG (SEQ ID NO: 164); TGSPAGLEADGSRQARVGS(SEQ ID NO: 162); TGPAGLGLEADGSRQARVG (SEQ ID NO: 166);TGRQARVGLEADGSPAGLG (SEQ ID NO: 167); TGSRQARVGPAGLEADGS (SEQ ID NO:168); and TGPAGLGSRQARVGLEADGS (SEQ ID NO:169); GPAGLGLEPDGSRQARVG (SEQID NO: 170); GGSGGGGIEPDIGGSGGS (SEQ ID NO: 171); GGSGGGGLEADTGGSGGS(SEQ ID NO: 172); GSIEPDIGS (SEQ ID NO: 173); GSLEADTGS (SEQ ID NO:174); GGSGGGGIEPDGGGSGGS (SEQ ID NO: 175); GGSGGGGIEPDVGGSGGS (SEQ IDNO: 176); GGSGGGGIEPDSGGSGGS (SEQ ID NO: 177); GGSGGGGIEPDTGGSGGS (SEQID NO: 178); GGGSLEPDGSGS (SEQ ID NO: 179); and GPAGLGLEADGSRQARVG (SEQID NO: 180), GGEGGGGSGGSGGGS (SEQ ID NO: 181); GSSAGSEAGGSGQAGVGS (SEQID NO: 182); GGSGGGGLEAEGSGGGGS (SEQ ID NO: 183); GGSGGGGIEPDPGGSGGS(SEQ ID NO: 184); TGGSGGGGIEPDIGGSGGS (SEQ ID NO: 185).

e. Anti-PD-1 VHH Domain

A constrained multispecific polypeptide construct of the disclosureincludes at least one PD-1 VHH domain from among any provided herein. Insome embodiments, the PD-1 VHH domain comprises the sequence of aminoacids set forth in any of SEQ ID NOS: 245-287, 294-299, and 312-315.

In particular embodiments, a constrained multispecific polypeptideconstruct contains at least two PD-1 VHH domain, such as any asdescribed. In some cases, at least one PD-1 VHH domain is positionedamino terminally relative to an Fc polypeptide of the heterodimeric Fcand at least one PD-1 VHH domain is positioned carboxy-terminallyrelative to VH or VL chain of the CD3 binding region. In some aspects,each of the two PD-1 VHH domain are the same.

In particular embodiments, a constrained multispecific polypeptideconstruct contains only PD-1 domain. In some cases, the PD-1 VHH domainis positioned amino terminally relative to an Fc polypeptide of theheterodimeric Fc. In some embodiments, the PD-1 VHH domain is positionedcarboxy-terminally relative to VH or VL chain of the CD3 binding region.

In some embodiments, the anti-PD-1 VHH domain is linked, directly orindirectly via a linker, to the Fc region and/or to the CD3 bindingregion. In some embodiments, linkage is via a linker. In someembodiments, the linker is a linking peptide (LP), which can include anyflexible or rigid linker as described. In some embodiments, the linkeris selected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQ IDNO: 244); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 245); GGSGGSGGSGGS, i.e.,(GGS)₄ (SEQ ID NO: 246); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO:247). In some embodiments, the linker is a flexible linker comprisingGlycine residues, such as, by way of non-limiting example, GG, GGG, GGGG(SEQ ID NO: 248), GGGGG (SEQ ID NO: 249), and GGGGGG (SEQ ID NO: 7). Insome embodiments, the linker includes a combination of a GS-linker and aGlycine linker.

f. Costimulatory Binding Domain

A multispecific polypeptide constructs of the present disclosure includeone or more co-stimulatory receptor binding region (CRBR) that binds acostimulatory receptor. In some embodiments, the one or more CRBR of theprovided multispecific polypeptide constructs bind a co-stimulatoryreceptor expressed on T cells. In some embodiments, the co-stimulatoryreceptor is upregulated, induced, or expressed on the surface of anactivated T cell. In some aspects, the CRBR binds a co-stimulatoryreceptor and stimulates the co-stimulatory receptor. In someembodiments, agonistic binding of the co-stimulatory receptor to theCRBR of the multispecific polypeptide induces downstream signaling inthe T cell to potentiate or enhance T cell activation or functionalitiesfollowing engagement of CD3. In some embodiments, the CRBR, orindependently each of the CRBRs, is an antibody or antigen bindingfragment, a natural cognate binding partner of the co-stimulatoryreceptor, an Anticalin (engineered lipocalin), a Darpin, a Fynomer, aCentyrin (engineered fibroneticin III domain), a cystine-knot domain, anAffilin, an Affibody, or an engineered CH3 domain.

In some embodiments, the CRBR, or independently each of the CRBRs, suchas the first CRBR and the second CRBRs, includes one or more copies ofan antibody or an antigen-binding fragment thereof. In some embodiments,the CRBR or independently each of the CRBRs, such as the firstantigen-binding domain and the second CRBRs, includes one or more copiesof an antibody or an antigen-binding fragment thereof selected from thegroup consisting of a Fab fragment, a F(ab′)₂ fragment, an Fv fragment,a scFv, a scAb, a dAb, a single domain heavy chain antibody, and asingle domain light chain antibody.

In some embodiments, the CRBR, or independently each of the CRBRs, suchas the first CRBR and the second CRBRs, is a single chain antibody. Insome examples, the single chain is an scFv, a scAb, a single domainheavy chain antibody, or a single domain light chain antibody.

In some embodiments, the CRBR, or independently each of the CRBRs, suchas the first CRBR and the second CRBR, includes one or more singledomain antibody (sdAb) fragments, for example V_(H)H, V_(NAR),engineered V_(H) or V_(K) domains. V_(H)Hs can be generated from naturalcamelid heavy chain only antibodies, genetically modified rodents thatproduce heavy chain only antibodies, or naïve/synthetic camelid orhumanized camelid single domain antibody libraries. V_(NAR)s can begenerated from cartilaginous fish heavy chain only antibodies. Variousmethods have been implemented to generate monomeric sdAbs fromconventionally heterodimeric V_(H) and V_(K) domains, includinginterface engineering and selection of specific germline families.

In some embodiments, the CRBR, or independently each of the CRBRs suchas the first CRBR and/or the second CRBR, of the multispecificpolypeptide constructs contains at least one sdAb or an scFv that bindsa costimulatory receptor. In some embodiments, the at least one scFv orsdAb that binds a costimulatory receptor is positioned amino-terminallyrelative to the Fc region and/or carboxy-terminally relative to the CD3binding region of the multispecific polypeptide construct. In someembodiments, the multispecific polypeptide construct contains only onescFv or sdAb that binds to a costimulatory receptor, which can bepositioned either amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region. In someembodiments, the multispecific polypeptide construct contains two scFvor sdAb that bind to a costimulatory receptor, positionedamino-terminally relative to the Fc region and/or carboxy-terminallyrelative to the CD3 binding region.

In some embodiments, the multispecific polypeptide construct is formedfrom or includes two polypeptides, including a first polypeptidecomprising a first Fc polypeptide of a heterodimeric Fc region, alinker, a VH domain of an anti-CD3 antibody or antigen binding fragment(e.g. Fv), and an scFv or sdAb that binds to a costimulatory receptor;and a second polypeptide comprising a second Fc polypeptide of theheterodimeric Fc region, the linker, a VL domain of the anti-CD3antibody or antigen binding fragment (e.g. Fv) and, optionally, another,the same or different, scFv or sdAb that binds to a costimulatoryreceptor. The scFv or sdAb that binds the costimulatory receptor can bepositioned amino terminally relative to an Fc polypeptide of theheterodimeric Fc and/or carboxy-terminally relative to a VH or VL chainof the CD3 binding region. At least one of the first and/or secondpolypeptide of the multispecific polypeptide construct also includes anantigen binding domain that binds a TAA or a chain thereof as describedin Section II.4. In some embodiments, the antigen binding domain thatbinds a TAA is a scFv or sdAb and is included as part of the firstand/or second polypeptide of the multispecific polypeptide construct. Insome embodiments, the antigen binding domain that binds a TAA is a Fab,and the multispecific polypeptide construct is additionally formed froma third polypeptide where at least the first and second polypeptideinclude a chain of the Fab that binds TAA (e.g. VH-CH1 or VL-CL of aFab) and the third polypeptide contains the other chain of the Fab thatbinds TAA (e.g. the other of VH-CH1 or VL-CL of a Fab).

In some embodiments, the CRBR or independently each of the CRBRs, suchas the first CRBR and/or the second CRBRs, contains more than one chain.In some embodiments, the CRBR or independently each of the CRBRs, suchas the first CRBR and/or the second CRBRs, of the multispecificpolypeptide constructs contains VH and VL sequences assembled as FABs.

In some embodiments, the CRBR antigen binding domain or independentlyeach of the CRBR antigen binding domains, such as the firstantigen-binding domain and/or the second antigen binding domains, of themultispecific polypeptide constructs contains a VH-CH1 (Fd) and a VL-CLof a Fab antibody that binds a costimulatory receptor. In someembodiments, the Fab antibody containing a VH-CH1 (Fd) and a VL-CL ispositioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct. In some embodiments, themultispecific polypeptide construct contains only one Fab antibody,containing a VH-CH1 (Fd) or VL-CL, that binds to a costimulatoryreceptor, which can be positioned either amino-terminally relative tothe Fc region and/or carboxy-terminally relative to the CD3 bindingregion. In some embodiments, the multispecific polypeptide constructcontains two Fab antibody fragments, each containing a VH-CH1 (Fd) andVL-CL, that binds to a costimulatory receptor, in which one ispositioned amino-terminally relative to the Fc region and the other ispositioned carboxy-terminally relative to the CD3 binding region.

In some embodiments, the multispecific polypeptide construct is formedfrom or includes three or more polypeptides, including a firstpolypeptide comprising a first Fc polypeptide of a heterodimeric Fcregion, a linker and a VH-CH1 (Fd) or VL-CL of a Fab antibody fragmentthat binds to a costimulatory receptor; a second polypeptide comprisinga second Fc polypeptide of the heterodimeric Fc region, the linker and,optionally, the same VH-CH1 (Fd) or VL-CL of the Fab antibody fragmentthat binds to a costimulatory receptor, and a third polypeptidecomprising the other of the VH-CH1 (Fd) or VL-CL of the Fab antibodyfragment that binds to the costimulatory receptor. The first, secondand/or third polypeptide of the multispecific polypeptide construct alsocan include a PD-1 VHH domain, such as any as described.

In some embodiments, the CRBR, or independently each of the CRBRs, is orincludes a natural (native) cognate binding partner of theco-stimulatory receptor (e.g. a natural ligand), or a variant thereofthat exhibits binding activity to the co-stimulatory receptor.

In some embodiments, the one or more CRBR of the provided multispecificpolypeptide constructs bind a co-stimulatory receptor expressed on Tcells. In some embodiments, there are more than one CRBR that binds to acostimulatory receptor and each of the CRBRs, such as the first CRBR andthe second CRBR, bind the same co-stimulatory receptor. In someembodiments, each of the CRBRs, such as the first CRBR and the CRBRs,bind a different co-stimulatory receptor. In some embodiments, each ofthe CRBRs, such as the first CRBR and the second CRBR bind a differentepitope on the same co-stimulatory receptor. In some embodiments, eachof the CRBRs, such as the first antigen-CRBR and the CRBR, bind the sameepitope on the same co-stimulatory receptor.

In some embodiments, the CRBR, or independently each of the CRBRs thatbinds a co-stimulatory receptor results in monovalent, bivalent,trivalent, or tetravalent binding to the co-stimulatory receptor.

In some embodiments, the co-stimulatory receptor is expressed on Tcells, such as primary T cells obtained from a subject. In someembodiments, the co-stimulatory receptor is expressed on human T cells,such as primary human T cells obtained from a human subject.

In some embodiments, the co-stimulatory receptor is a member of thetumor necrosis factor (TNF) receptor family. In some embodiments, thecostimulatory receptor is a member of the immunoglobulin superfamily(IgSF). In some embodiments, the costimulatory receptor is a member ofthe B7 family of receptors.

In some embodiments, the co-stimulatory receptor is selected from thegroup consisting of 41BB (CD137), OX40 (CD134), CD27,glucocorticoid-induced TNFR-related protein (GITR), CD28, ICOS, CD40,B-cell activating factor receptor (BAFF-R), B-cell maturation antigen(BCMA), Transmembrane activator and CAML interactor (TACI), and NKG2D.In some embodiments, the co-stimulatory receptor is selected from 41BB,OX40, GITR, ICOS, or CD28. In some embodiments, the co-stimulatoryreceptor is selected from 41BB, OX40, or GITR.

In some embodiments, the costimulatory receptor is 41BB. In someembodiments, the costimulatory receptor is OX40. In some embodiments,the costimulatory receptor is GITR. In some embodiments, thecostimulatory receptor is ICOS. In some embodiments, the costimulatoryreceptor is CD28.

In some embodiments, the CRBR of the multispecific polypeptide is orcomprises an agonistic binding molecule to the co-stimulatory receptor.The CRBR can bind to the co-stimulatory receptor and initiate, induce,or stimulate a reaction or activity that is similar to or the same asthat initiated, induced, or stimulated by the receptor's natural ligand.In some aspects, the binding of the CRBR to the co-stimulatory receptorinduces or stimulates a downstream signal that is more than 5%, morethan 10%, more than 20%, more than 30%, more than 40%, more than 50%,more than 60%, more than 70%, more than 80%, more than 90%, or more than100% of the signal that is initiated, induced, or stimulated by thereceptor's natural ligand.

In some embodiments, the one or more CRBR is an antibody or fragmentthereof that binds to the co-stimulatory receptor 41BB (CD137), OX40(CD134), CD27, glucocorticoid-induced TNFR-related protein (GITR), CD28,ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cellmaturation antigen (BCMA). In some embodiments, the one or more CRBR isan antibody or fragment thereof that binds to the co-stimulatoryreceptor 41BB, OX40, GITR, ICOS, or CD28. In some embodiments, the oneor more CRBR is an antibody or fragment thereof that binds to theco-stimulatory receptor 41BB, OX40, or GITR. Exemplary polypeptides forbinding 41BB, OX40 and GITR are described in PCT publication. No.WO2017123650, WO2017123673, and WO2017015623, respectively. In someembodiments, the one or more CRBR is a single domain antibody (sdAb)that binds the co-stimulatory receptor, such as those described in PCTpublication. No. WO2017123650, WO2017123673, and WO2017015623.

In some examples, the co-stimulatory receptor binding region (CRBR)binds or comprises a natural cognate binding partner of 41BB (CD137),OX40 (CD134), CD27, glucocorticoid-induced TNFR-related protein (GITR),CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cellmaturation antigen (BCMA), Transmembrane activator and CAML interactor(TACI), NKG2D. In some embodiments, the natural cognate binding partneris selected from 41BB ligand (41BBL), OX40L (CD252), CD70, GITRLigand/TNFSF18, CD80 (B7-1), CD86 (B7-2), ICOS Ligand (ICOSL), CD154(CD40L), B-cell activating factor (BAFF), A proliferation-inducingligand (APRIL), NKG2D ligands, or a functional fragment thereof.

Exemplary sequences of CRBRs are set forth in Table 4.

In some embodiments, at least one CRBR, or independently each CRBR,binds the co-stimulatory receptor 41BB. In some examples, the CRBR is orcontains an antibody or antigen binding fragment specific to or thatbinds 41BB, such as a sdAb or fragments containing a VH and VL (e.g.scFv). In some embodiments, at least on CRBR, or independently eachCRBR, is a natural ligand of 41BB or is a functional binding fragmentthereof. In some embodiments, at least one CRBR, or independently eachCRBR, is an anticalin. Exemplary of such 41BB-binding CRBRs are setforth in any of SEQ ID NOS: 186-210. In some embodiments, the 41BBbinding CRBR contains a VH set forth in any of SEQ ID NOS: 187, 189 and191, and a VL set forth in any of SEQ ID NO: 188, 190, or 192. TheCRBRs, or independently each CRBR, in a provided multispecificpolypeptide construct can have at least 85%, 85%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyof the foregoing SEQ ID Nos and bind 41BB.

In some embodiments, at least one CRBR, or independently each CRBR,binds the co-stimulatory receptor OX40. In some examples, the CRBR is orcontains an antibody or antigen binding fragment specific to or thatbinds OX40, such as a sdAb or fragments containing a VH and VL (e.g.scFv). In some embodiments, at least on CRBR, or independently eachCRBR, is a natural ligand of OX40 or is a functional binding fragmentthereof. Exemplary of such OX40-binding CRBRs are set forth in any ofSEQ ID NOS: 211-220. In some embodiments, the OX40-binding CRBR containsan VH set forth in any of SEQ ID NOS: 216 and 218, and a VL set forth inany of SEQ ID NO: 217 and 219. The CRBRs, or independently each CRBR, ina provided multispecific polypeptide construct can have at least 85%,85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to any of the foregoing SEQ ID Nos and bind OX40.

In some embodiments, at least one CRBR, or independently each CRBR,binds the co-stimulatory receptor GITR. In some examples, the CRBR is orcontains an antibody or antigen binding fragment specific to or thatbinds GITR, such as a sdAb or fragments containing a VH and VL (e.g.scFv). In some embodiments, at least one CRBR, or independently eachCRBR, is a natural ligand of GITR or is a functional binding fragmentthereof. Exemplary of such GITR-binding CRBRs are set forth in any ofSEQ ID NOS: 221-230. In some embodiments, the GITR binding CRBR containsa VH set forth in any of SEQ ID NOS: 222, 224, 226, and 228 and a VL setforth in any of SEQ ID NO: 223, 225, 227, and 229. The CRBRs, orindependently each CRBR, in a provided multispecific polypeptideconstruct can have at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the foregoingSEQ ID Nos and bind GITR.

In some embodiments, at least one CRBR, or independently each CRBR,binds the co-stimulatory receptor CD27. In some examples, the CRBR is orcontains an antibody or antigen binding fragment specific to or thatbinds CD27, such as a sdAb or fragments containing a VH and VL (e.g.scFv). In some embodiments, at least one CRBR, or independently eachCRBR, is a natural ligand of CD27 or is a functional binding fragmentthereof. Exemplary of such CD27-binding CRBRs are set forth in any ofSEQ ID NOS: 231. In some embodiments, the CD27 binding CRBR contains aVH set forth SEQ ID NO: 232 and a VL set forth in SEQ ID NO: 233. TheCRBRs, or independently each CRBR, in a provided multispecificpolypeptide construct can have at least 85%, 85%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyof the foregoing SEQ ID Nos and bind CD27.

In some embodiments, at least one CRBR, or independently each CRBR,binds the co-stimulatory receptor ICOS. In some examples, the CRBR is orcontains an antibody or antigen binding fragment specific to or thatbinds ICOS, such as a sdAb or fragments containing a VH and VL (e.g.scFv). In some embodiments, at least one CRBR, or independently eachCRBR, is a natural ligand of ICOS or is a functional binding fragmentthereof. An exemplary ICOS-binding CRBR sequence is set forth in SEQ IDNO: 234.

In some embodiments, at least one CRBR, or independently each CRBR,binds the co-stimulatory receptor CD28. In some examples, the CRBR is orcontains an antibody or antigen binding fragment specific to or thatbinds CD28, such as a sdAb or fragments containing a VH and VL (e.g.scFv). In some embodiments, at least one CRBR, or independently eachCRBR, is a natural ligand of CD28 or is a functional binding fragmentthereof. An exemplary CD28-binding CRBR sequence is set forth in SEQ TDNO: 235.

TABLE 4 Exemplary CRBR Sequences CRBR Format Reference SEQ ID NO 41BBbinding CRBR Sequences 41BBL Natural Ligand UniProt accession no. P41273186 PF-05082566 VH US 2012/0237498 (SEQ ID NO: 187 43) VL US2012/0237498 (SEQ ID NO: 188 45) BMS663513 VH WO 2005/035584 (SEQ ID NO:9) 189 VL WO 2005/035584 (SEQ ID NO: 6) 190 MSB7 VH US 2017/0226215 (SEQID NO: 191 138) VL US 2017/0226215 (SEQ ID NO: 192 28) 41BB AnticalinAnticalin WO 2016/177762 (SEQ ID NO: 12) 193 41BB Anticalin Anticalin WO2016/177762 (SEQ ID NO: 13) 194 41BB Anticalin Anticalin WO 2016/177762(SEQ ID NO: 14) 195 41BB Anticalin Anticalin WO 2016/177762 (SEQ ID NO:15) 196 41BB Anticalin Anticalin WO 2016/177762 (SEQ ID NO: 16) 197 41BBAnticalin Anticalin WO 2016/177762 (SEQ ID NO: 17) 198 41BB AnticalinAnticalin WO 2016/177762 (SEQ ID NO: 18) 199 41BB Anticalin Anticalin WO2016/177762 SEQ ID NO: 19) 200 41BB Anticalin Anticalin WO 2016/177762(SEQ ID NO: 20) 200 71-254 of human 41BB ligand WO 2017/167672 (SEQ IDNO: 3) 201 41BBL 85-254 of human 41BB ligand WO 2017/167672 (SEQ ID NO:4) 202 41BBL 80-254 of human 41BB ligand WO 2017/167672 (SEQ ID NO: 5)203 41BBL 52-254 of human 41BB ligand WO 2017/167672 (SEQ ID NO: 6) 20441BBL 71-248 of human 41BB ligand WO 2017/167672 (SEQ ID NO: 7) 20641BBL 85-248 of human 41BB ligand WO 2017/167672 (SEQ ID NO: 8 207 41BBL80-248 of human 41BB ligand WO 2017/167672 (SEQ ID NO: 9) 208 41BBL52-248 of human 41BB ligand WO 2017/167672 (SEQ ID NO: 10) 209 41BBL41BB sdAb sdAb US 2017/0198050 210 OX40-binding CRBR Sequences OX40ligand Natural Ligand UniProt accession no. P23510 211 OX40 ligandNatural Ligand U.S. Pat. No. 7,959,925 (SEQ ID NO: 2) 212 human OX40L:Natural Ligand WO 2017/167672 (SEQ ID NO: 213 51-183 11) Human Ox40L:Natural Ligand WO 2017/167672 (SEQ ID NO: 214 51-183 N90D 12) HumanOX40L: Natural Ligand WO 2017/167672 (SEQ ID NO: 215 52-183 13) 1A07 VHUS 2015/0307617 (SEQ ID NO: 216 56) VL US 2015/0307617 (SEQ ID NO: 21759) 1949 VH WO 2016/179517 (SEQ ID NO: 218 16) VL W02016/179517 2191D10v1 sdAb U.S. Pat. No. 9,006,399 220 GITR-binding CRBR Sequences GITRligand Natural Ligand UniProt no. Q9UNG2 221 36E5 VH US 2014/0348841(SEQ ID NO: 222 104) VL US 2014/0348841 (SEQ ID NO: 223 105) TRX-518 VHUS 2013/0183321 (SEQ ID NO: 224 54) VL US 2013/0183321 (SEQ ID NO: 22544) 5H7v2 VH US 2015/0064204 (SEQ ID NO: 226 282) VL US 2015/0064204(SEQ ID NO: 227 134) 41G5v2 VH US 2015/0064204 (SEQ ID NO: 228 312) VLUS 2015/0064204 (SEQ ID NO: 229 124) C06v3 sdAb US 2017/0022284 (SEQ IDNO: 230 59) CD27-binding CRBR Sequences CD70-ECD Natural Ligand UniProtno. P32970 231 1F5 VH US 2011/0274685 232 VL US 2011/0274685 233ICOS-binding CRBR Sequences ICOS sdAb sdAb 234 CD28 binding CRBRSequences CD28 sdAb sdAb 235

In some embodiments, the one or more CRBR is linked, directly orindirectly via a linker, to the Fc region and/or to the CD3 bindingregion. In some embodiments, linkage is via a linker. In someembodiments, the linker is a linking peptide (LP), which can include anyflexible or rigid linker as described herein, although generally thepeptide linking the CRBR or regions is not a cleavable liker.

In some embodiments, the multispecific polypeptide construct comprises alinking peptide (LP) between the CRBR and the Fc region. In someembodiments, the multispecific polypeptide construct comprises a linkingpeptide (LP) between the CD3 binding region and the CRBR.

3. NK Recruitment

In some embodiments, the PD-1-binding polypeptide is a bispecificconstruct that is or comprises at least one PD-1 VHH domain providedherein and at least one additional binding molecule capable of bindingto a surface molecule expressed on a Natural Killer (NK) cells and/orrecruiting NK cells. In particular aspects, the multispecific constructis bispecific for PD-1 and the NK cell surface molecule. In someembodiments, the surface molecule is CD16 (FcγRIII). Specifically, aprovided bispecific PD-1-binding polypeptide is capable of specificallybinding an NK activating receptor expressed on a human NK cells cell,such as human CD16a.

CD16, a low affinity receptor for the Fc portion of some IgGs known tobe involved in antibody-dependent cellular cytotoxicity (ADCC), is thebest-characterized membrane receptor responsible for triggering oftarget cell lysis by NK cells (Mandelboim et al., 1999, PNAS96:5640-5644). Generally, a large majority (approximately 90%) of humanNK cells express CD56 at low density (CD56dim) and FcγRIII (CD16) at ahigh level (Cooper et al., 2001, Trends Immunol. 22:633-640). HumanFcγRIII exists as two isoforms, CD16a (FcγRIIIA) and CD16b (FcγRIIIB),that share 96% sequence identity in their extracellularimmunoglobulin-binding regions (van de Winkel and Capel, 1993, Immunol.Today 14(5):215-221). In particular embodiments, the additional bindingmolecule is capable of specifically binding CD16a.

CD16a is expressed on macrophages, mast cells, and NK cells as atransmembrane receptor. On NK cells, the alpha chain of CD16a associateswith the immunoreceptor tyrosine-based activation motif (ITAM)containing FcεRI γ-chain and/or the T-cell receptor (TCR)/CD3 ζ-chain tomediate signaling (Wirthmueller et al., 1992, J. Exp. Med.175:1381-1390). The interaction of CD16a with different combinations ofhomo- and hetero-dimers of the γ and ζ chains has been observed in NKcells, indicating the ability to mediate signaling via differentsignaling pathways via variations of the CD16a complex in NK cells(Anderson et al., 1990, PNAS 87(6):2274-2278; Ackerly et al., 1992, Int.J. Cancer Suppl. 7:11-14). FcγR-expressing effector cells have beenshown to be involved in destroying tumor cells via ADCC. For example,engagement of CD16a, such as with an agonist binding molecule capable ofspecifically binding CD16a can result in activating of NK cellsexpressing CD16a, thereby eliciting a biological response, in particulara signaling response. In some cases, the binding molecule is capable oftriggering cell killing, in a manner analogous to antibody-dependentcellular cytotoxicity (ADCC), by virtue of its binding to such cells.

In particular example, PD-1-binding polypeptides include bispecificmolecules that can specifically bind to PD-1 and to CD16a may target NKcells to cells bearing such antigen, so that the cell bearing theantigen may be eradicated via NK cell mediated cell killing. Forexample, a binding molecule that specifically binds PD-1 expressed on atumor cell may target NK-cells to the tumor cell. In some cases,activation of the NK cell caused by the binding molecule binding toCD16a can lead to killing of the tumor cells.

In some embodiments, the additional binding domain specific to anactivating NK cell receptor, such as CD16a, is an antigen-bindingfragment selected from a Fab fragment, a F(ab′)₂ fragment, an Fvfragment, a scFv, disulfide stabilized Fv fragment (dsFv), a scAb, adAb, a single domain heavy chain antibody (VHH), or a single domainlight chain antibody. In some embodiments, the additional binding domainis monovalent for binding the activating T NK cell receptor, such asCD16a.

In some cases, the additional binding domain recognizes CD16a. In someembodiments, the anti-CD16a binding domain includes one or more copiesof an anti-CD16a Fab fragment, an anti-CD16a F(ab′)₂ fragment, ananti-CD16a Fv fragment, an anti-CD16a scFv, an anti-CD16a dsFv, ananti-CD16a scAb, an anti-CD16a dAb, an anti-CD16a single domain heavychain antibody (VHH), and an anti-CD16a single domain light chainantibody. In some embodiments, the anti-CD16a binding domain ismonovalent for binding CD16a. In some embodiments, the BH73-bindingpolypeptide is a bispecific construct that binds BH73 and agonizes theactivity of CD16a.

Antibodies and antigen-binding fragments thereof specific for CD16a areknown and include, for example, NM3E2 (McCall et al. (1999) Mol.Immunol., 36:433-045. Other anti-CD16a antibodies also can be used inthe constructs provided herein, including any described in publishedU.S. patent application No. US1060280795; U.S. Pat. No. 9,701,750; Beharet al. (2008) Protein Eng Des Sel. 21:1-10; Arndt et al., (1999) Blood94:2562-2568. In particular examples, the anti-CD16a is an anti-CD16ascFv. In some embodiments, the anti-CD16a is an anti-CD16a antibodyincluded in a TandAb molecule (see e.g. Reush et al. (2014) Mabs,6:727-738). In some aspects, the anti-CD16a is an anti-CD16a or antigenbinding fragment, such as an scFv, described in U.S. Pat. No. 9,035,026.

The provided bispecific constructs can be formatted in any of a numberof formats containing the at least one PD-1 VHH domain and the at leastone additional domain specific to an activating NK cell receptor, suchas a CD16a-binding domain.

In one embodiment, the bispecific construct is a bispecificsingle-domain antibody-linked Fab (S-Fab) containing at least one PD-1VHH domain as described linked, directly or indirectly to a Fab antigenbinding fragment specific to an NK cell activating receptor, e.g. CD16a,such as an anti-CD16a Fab. In some embodiments, the PD-1 VHH domain islinked to the C-terminus of the VH or VL chain of an anti-C16a Fab. Insome embodiments, the S-Fab can be further modified, such as byconjugation with polyethylene glycol (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, proteins (such as albumin),polyglutamic acid or PASylation (Pan et al. (2018) International Journalof Nanomedicine, 2018:3189-3201).

In another embodiment, the bispecific construct is a scFv-single domainantibody in which the construct contains at least one PD-1 VHH asdescribed linked, directly or indirectly, to an scFv containing a VH anda VL of an antigen binding domain specific to an NK cell activatingreceptor, e.g. CD16a. The scFv against an NK cell activating receptor,e.g. anti-CD16a scFv, can contain any of the VH and VL sequences asdescribed. In some embodiments, the VHH domain and the scFv areconnected by a linker, such as a peptide linker. In some embodiments,the peptide linker can be a peptide linker as described herein. In someembodiments, the VHH domain and the scFv are each connected, optionallythrough a hinge region or a linker (e.g. peptide linker), to an Fcregion, such as an N-terminus of an Fc region. The Fc region can be anydescribed herein, such as a human Fc region or a variant thereof, e.g. ahuman IgG1 Fc region or a variant thereof. In particular examples, theFc region is formed by variant Fc domains, e.g. variant human IgG1domains, that are mutated or modified to promote heterodimerization inwhich different polypeptides can be dimerized to yield a heterodimer.

In a further embodiment, the antigen binding domain specific to an NKcell activating receptor, e.g. CD16a, is a single domain antibody, suchas is a VHH domain that specifically binds to CD16a. Single domainantibodies, including VHH domains that bind to CD16a are known, see e.g.published U.S. patent application No. US20160280795. In such aspects, abispecific construct provided herein can include at least one PD-1 VHHdomain and at least one CD16a VHH domain. For formatting the constructs,in some cases, each VHH domain is connected, optionally through a hingeregion or linker (e.g. peptide linker), to an Fc region, such as anN-terminus of an Fc region. The Fc region can be any described herein,such as a human Fc region or a variant thereof, e.g. a human IgG1 Fcregion or a variant thereof. In particular examples, the Fc region isformed by variant Fc domains, e.g. variant human IgG1 domains, that aremutated or modified to promote heterodimerization in which differentpolypeptides can be dimerized to yield a heterodimer.

In the above embodiments, exemplary modifications of an Fc region topromote heterodimerization are known, including any as described below,e.g. Table 1. In some embodiments, one Fc polypeptide of a heterodimericFc comprises the sequence of amino acids set forth in any of SEQ ID NOS:103, 107, 115, or 117, and the other Fc polypeptide of the heterodimericFc contains the sequence of amino acids set forth in any of SEQ ID NOS:104, 108, 111, 113, 119 or 121. In some embodiments, one Fc polypeptideof a heterodimeric Fc comprises the sequence of amino acids set forth inany of SEQ ID NOS: 105, 109, 116 or 118 and the other Fc polypeptide ofthe heterodimeric Fc comprises the sequence of amino acids set forth inany of SEQ ID NOS: 106, 110, 112, 114, 120 or 122.

4. Cytokine Fusion and/or Cytokine Receptor Targeting

In some embodiments, the PD-1-binding polypeptide is a multispecificpolypeptide construct that is a cytokine-antibody fusion protein (alsocalled a PD-1 VHH-cytokine fusion). In some aspects, at least one PD-1VHH domain provided herein is linked, directly or indirectly, to atleast one cytokine, such as to an interferon. In particular embodiments,the cytokine is an interferon capable of exhibiting anti-proliferativeactivity, apoptotic activity and/or anti-viral activity. In someembodiments, the interferon of a PD-1 VHH-cytokine fusion providedherein is capable of binding to a receptor composed of IFNAR1 and/or 2.Any of a variety of assays can be used to assess the effect of suchfusion proteins on binding IFNAR1 and/or 2, reducing or decreasing thegrowth rather and/or proliferation rate of a cancer cell, reducing tumorsize, eliminating tumors or inducing the death of a cancer cell (e.g.via apoptosis). Such assays in include in vitro assays with variouscancer cell lines known to express PD-1 or in vivo assays employinganimal tumor models.

In some embodiments, the interferon is a type I interferon, such as ahuman type I interferon or a variant thereof. In some aspects, the humantype I interferon is a variant that is a truncated human type Iinterferon or a human mutant type I interferon. In some embodiments, thetype I interferon or variant thereof is a wild-type human IFN-alpha(IFN-alpha; alpha2 and natural higher affinity variants such as alpha14), interferon beta (IFN-beta) as well as mutants and/or truncatedforms thereof. In some embodiments, the interferon is a type IIinterferon, such as a human type II interferon or a variant thereof. Insome aspects, the human type II interferon is a variant that is atruncated human type II interferon or a human mutant type II interferon.In some embodiments, the type II interferon or variant thereof is awild-type human interferon gamma (IFN-gamma) as well as mutants and/ortruncated forms thereof. In some embodiments, the providedcytokine-antibody fusion proteins can be used to inhibit the growthand/or proliferation of target cells (e.g. cancer cells) that express oroverexpress PD-1.

In some embodiments, the PD-1 VHH-cytokine fusion protein is similar informat to any as described in International PCT published applicationNo. WO2014194100; U.S. Pat. No. 9,803,021; Valedkarimi et al. (2017)Biomed Pharmacother., 95:731-742; or Young et al. (2014) Semin Oncol.,41:623-636.

In particular embodiments, the interferon, e.g. a type I interferon,such as a human type I interferon (e.g. IFN-alpha, IFN-beta, orIFN-gamma) is one that possesses the endogenous binding affinity and/oractivity of the native or wild-type interferon, preferably at a level ofat least 60%, or of at least or at least about 80%, such as at least90%, 95%, 98%, 99%, 100%, or a level greater than the native wild-typeinterferon (in its isolated form).

Interferons and interferon mutants are a well-known and wellcharacterized group of cytokines (see e.g., WO 2002/095067; WO2002/079249; WO 2002/101048; WO 2002/095067; WO 2002/083733; WO2002/086156; WO 2002/083733; WO 2003/000896; WO 2002/101048; WO2002/079249; WO 2003/000896; WO 2004/022593; WO2004/022747; WO2003/023032; WO 2004/022593 and also in Kim et al. (2003) Cancer Lett.189(2): 183-188; Hussain et a/. (2000) J. Interferon Cytokine Res.20(9): 763-768; Hussain et al. (1998) J. Interferon Cytokine Res. 18(7):469-477; Nyman et al. (1988) Biochem. J. 329 (Pt 2): 295-302; Golovlevaet al. (1997) J. Interferon Cytokine Res. 17(10): 637-645; Hussain etal. (1997) J. Interferon Cytokine Res. 17(9): 559-566; Golovleva et al.(1997) Hum. Hered. 47(4): 185-188; Kita et al. (1991) J. InterferonCytokine Res. 17(3): 135-140; Golovleva et al. (1996) Am. J. Hum. Genet.59(3): 570-578; Hussain et al. (1996) J. Interferon Cytokine Res. 16(7):523-529; Linge et al. (1995) Biochim Biophys Acta. Any of such can beused in the provided cytokine-antibody fusion proteins.

In some embodiments, the interferon is a human type I interferon.Alleles of the human interferon family of genes/proteins are known, seee.g. Pestka (10983) Arch Biochem Biophys., 221:1-37; Diaz et al. (1994)Genomics, 22:540-52; Pestka (1986) Meth. Enzymol, 199: 3-4; and Krauseet al. (2000) J. Biol. Chem., 275:22995-3004.

In some embodiments, the interferon is a full-length IFN-alpha (e.g.human IFN-alpha), a full-length IFN-beta (e.g. human IFN-beta) or afull-length IFN-gamma (e.g. human IFN-gamma). In some embodiments, theinterferon is a biologically active truncated IFN-alpha (e.g. humanIFN-alpha), a biologically active truncated IFN-beta (e.g. humanIFN-beta) or a biologically active truncated IFN-gamma (e.g. humanIFN-gamma). In some embodiments, a biologically active truncatedinterferon contains a contiguous sequence of amino acids of a wild-typeor native interferon that is truncated at the N- and/or C-terminus andcomprises a length that is at least or at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90% or more the length of the native orwild-type interferon. Any of a variety of standard assays for assessingbiological activity of an interferon can be used. For example, IFN-alphaactivity can be assayed by measuring antiviral activity against aparticular test virus. Kits for assaying for IFN-alpha activity arecommercially available (see, e.g., ILITE™ alphabeta kit by Neutekbio,Ireland). In some aspects, the IFN-alpha is an IFN-a2a (e.g. Acc. No.CAA23805), IFN-a-c (Acc. No. P01566), IFN-a-d (Acc. No. AAB59403);IFNa-5 (Acc. No. CAA26702); IFNa-6 (Acc. No. AA26704); IFNa-4 (Acc. No.NP_066546); IFNa-4b (Acc. No. CAA26701); IFNa-I (Acc. No. AAA52725);IFNa-J (Acc. No. CAA23792); IFNa-H (Acc. No. CAA23794); IFNa-F (Acc. No.AAA52718); IFNa-7 (Acc. No. CAA26903), or is a biologically activefragment thereof. In some aspects, the IFN-beta is IFN-beta set forth inAcc. No. AAC41702 or is a biologically active fragment thereof. In someaspects, the IFN-gamma is IFN-gamma set forth in Acc. No. P01579 or is abiologically active fragment thereof.

In some embodiments, a provided PD-1 VHH-cytokine fusion contains avariant or mutant interferon alpha 2 (IFNa2) is contemplated. Certainmutants include a mutation of the His at position 57, and/or the E atposition 58, and/or the Q at position 61. In certain embodiments themutants include the mutation H57Y, and/or E58N, and/or Q61S. In certainembodiments the mutants include a mutated IFNa2 having the mutationsH57Y, E58N, and Q61S (YNS) (see, e.g., Kalie et al. (2007) J. Biol.Chem., 282: 11602-11611). In other embodiments mutants include amutation of the His at position 57, and/or the E at position 58, and/orthe Q at position 61 to A (alanine). In certain embodiments the mutantsinclude a mutated IFNa2 having the mutations H57A, E58A, and Q61A (HEQ)(see, e.g., Jaitin et al. (2006) Mo/. Cellular Biol, 26(5): 1888-1897).In certain embodiments the mutant interferon comprises a mutation of Hisat position 57 to A, Y, or M, and/or a mutation of E at position 58 toA, or N, or D, or L, and/or a mutation of Q at position 61 to A, or S,or L, or D. In certain embodiments mutant include mutants of interferonalpha 8 (IFN-a8), such as variants with amino acid replacementcorresponding to R145 to V, I, or L, and/or A146 to N, or S, and/or M149to Y, e.g. R145V/A146N/M149Y), R145I/A146S/M149Y or R145L/A146S/M149Y(see, e.g, Yamamoto et. al, (2009) J. Interferon & cytokine Res, 29:161-170.

In some embodiments, a provided PD-1 VHH-cytokine fusion contains amutant or variant IFN-beta containing a serine substituted for thenaturally occurring cysteine at amino acid 17 (see, e.g., Hawkins et al.(1985) Cancer Res., 45, 5914-5920).

In some embodiments, a provided PD-1 VHH-cytokine fusion contains atruncated interferon. In one embodiment, a truncated interferon includesa human IFN-alpha with deletions of up to the first 15 amino-terminalamino acid residues and/or up to the last 10-13 carboxyl-terminal aminoacid residues, which has been shown to retain activity of the native orwild-type human IFN-alpha (see e.g. Ackerman (1984) Proc. Natl. Acad.Sci, USA, 81: 1045-1047). In some embodiments, a truncated humanIFN-alpha has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 carboxylterminal amino acid residues deleted and/or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 amino terminal amino acid residues deleted.

In some embodiments, a provided PD-1 VHH-cytokine fusion contains atruncated interferon, such as described in published U.S. patent appl.No. US2009/0025106. In some embodiments, a provided PD-1 VHH-cytokinefusion contains a truncated IFN-gamma containing N- and/or C-terminaldeletions, such as described in Lundell et al. (1991) Protein Neg.,4:335-341; Pan et al. (1987) Eur. J. Biochem., 166:145-149.

In some embodiments, the interferon, e.g. human interferon, is a mutantinterferon that is resistant to proteolysis compared to the unmodified,typically wild-type protein, see e.g. U.S. Pat. Nos. 7,998,469;8,052,964; 4,832,959 6,120,762; WO1992/008737; and EP219781.

In aspects of the provided PD-1 VHH-cytokine fusion proteins, theantibody and the cytokine, e.g. interferon, are attached directly or areattached indirectly via a linker, such as a peptide linker. Theattachment can be to the N- or C-terminus of the VHH domain, so long asthe attachment does not interfere with binding of the antibody to PD-1.Any linker, e.g. peptide linker, described herein can be used. In someembodiments, the linker is a GS-linker that comprises an amino acidsequence selected from the group consisting of GGSGGS, i.e., (GGS)₂ (SEQID NO: 1); GGSGGSGGS, i.e., (GGS)₃ (SEQ ID NO: 2); GGSGGSGGSGGS, i.e.,(GGS)₄ (SEQ ID NO: 3); and GGSGGSGGSGGSGGS, i.e., (GGS)₅ (SEQ ID NO: 4).In some embodiments, the linker is a flexible linker comprising Glycineresidues, such as, by way of non-limiting example, GG, GGG, GGGG (SEQ IDNO: 5), GGGGG (SEQ ID NO: 6), and GGGGGG (SEQ ID NO: 7). In someembodiments, the fusion proteins can include a combination of aGS-linker and a Glycine linker.

D. Engineered Cells

Provided herein are engineered cells that express any of the providedPD-1 binding molecules described herein. In particular example, theprovided PD-1 bind molecules are secreted from the cell. In someembodiments, the PD-1 binding molecule, e.g. containing an anti-PD-1 VHHdomain as provided herein, comprises a signal peptide, e.g., an antibodysignal peptide or other efficient signal sequence to get domains outsideof cell. When the PD-1 binding molecule comprises a signal peptide andis expressed by an engineered cell, the signal peptide causes theimmunomodulatory protein to be secreted by the engineered cell.Generally, the signal peptide, or a portion of the signal peptide, iscleaved from the binding molecule with secretion. The PD-1 bindingmolecule can be encoded by a nucleic acid (which can be part of anexpression vector). In some embodiments, the PD-1 binding molecule isexpressed and secreted by a cell (such as an immune cell, for example aprimary immune cell).

In some embodiments, provided engineered cells further contain achimeric antigen receptors (CARs). CARs are synthetic receptorstypically containing an extracellular targeting/binding moiety that isassociated with one or more signaling domains in a single fusionmolecule, and that is expressed on the surface of a cell, such as a Tcell. Thus, CARs combine antigen-specificity and T cell activatingproperties in a single fusion molecule. First generation CARs typicallyincluded the cytoplasmic region of the CD3zeta or Fc 1 receptor 7 chainas their signaling domain. First generation CARs have been tested inphase I clinical studies in patients with ovarian cancer, renal cancer,lymphoma, and neuroblastoma, where they have induced modest responses(reviewed in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009).Second generation CARs, which contain the signaling domains of acostimulatory molecule, such as CD28, and CD3zeta, provide dualsignaling to direct combined activating and co-stimulatory signals.Third generation CARs are more complex with three or more signalingdomains (reviewed in Sadelain et al., Cancer Discovery (3), 388-398,2013 and Dotti et al, Immuno. Rev, 257 (1), 1-36, 2014).

In some embodiments, a CAR contains an extracellular domain comprisingone or more antigen binding domain specific to a tumor antigen. In someaspects, the tumor antigen and/or antigen binding domain specific to aTAA can be any as described herein. CAR constructs include anextracellular domain containing the one or more extracellular antigenbinding domain, a transmembrane domain and an intracellular signalingregion. In some cases, the extracellular antigen binding domain is anscFv or a single domain antibody (VHH). In general, the extracellularantigen binding domain which form the antigen binding unit of the CAR“binds” or is “capable of binding”, i.e. targets, a target antigen withsufficient affinity such the CAR is useful in therapy in targeting acell or tissue expressing the target antigen.

The transmembrane domain of a CAR is a domain that typically crosses oris capable of crossing or spanning the plasma membrane and is connected,directly or indirectly (e.g. via a spacer, such as an immunoglobulinhinge sequence) to the extracellular antigen binding domain and theendoplasmic portion containing the intracellular signaling domain. Inone embodiment, the transmembrane domain of the CAR is a transmembraneregion of a transmembrane protein (for example Type I transmembraneproteins), an artificial hydrophobic sequence or a combination thereof.In one embodiment, the transmembrane domain comprises the CD3zeta domainor CD28 transmembrane domain. Other transmembrane domains will beapparent to those of skill in the art and may be used in connection withembodiments of a CAR provided herein.

The intracellular signaling region of a CAR provided herein contains oneor more intracellular signaling domain that transmits a signal to a Tcell upon engagement of the antigen binding domain of the CAR, such asupon binding antigen. In some embodiments, the intracellular regioncontains an intracellular signaling domain that is or contains an ITAMsignaling domain. Exemplary intracellular signaling domains include, forexample, a signaling domain derived from ζ chain of the T-cell receptorcomplex or any of its homologs (e.g., η chain, FcsRIy and β chains, MB 1(Iga) chain, B29 (Ig) chain, etc.), human CD3zeta chain, CD3polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70,etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and othermolecules involved in T-cell transduction, such as CD2, CD5, OX40 andCD28. In particular embodiments, the intracellular signaling regioncontains an intracellular signaling domain derived from the human CD3zeta chain.

In some embodiments, the endodomain comprises at CD3-zeta signalingdomain. In some embodiments, the CD3-zeta signaling domain comprises thesequence of amino acids set forth in SEQ ID NO: 236 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity toSEQ ID NO: 236 and retains the activity of T cell signaling.

In some embodiments, the intracellular signaling region of a CAR canfurther contain an intracellular signaling domain derived from acostimulatory molecule. In such examples, such a signaling domain mayenhance CAR-T cell activity, such as via enhancement of proliferation,survival and/or development of memory cells, after antigen specificengagement, for example, compared to a CAR that only contains an ITAMcontaining signaling domain, e.g. CD3 zeta. In some embodiments, theco-stimulatory domain is a functional signaling domain obtained from aprotein selected from: CD28, CD137 (4-IBB), CD134 (OX40), DapIO, CD27,CD2, CD5, ICAM-1, LFA-1 (CD1 la/CD18), Lck, TNFR-I, TNFR-II, Fas, CD30,CD40 or combinations thereof. In particular embodiments, thecostimulatory signaling domain is derived or obtained from a humanprotein. In some aspects, the costimulatory signaling domain is derivedor obtained from human CD28 or human CD137 (4-IBB).

In some embodiments, the costimulatory signaling domain is a derivedfrom CD28 or 4-1BB and comprises the sequence of amino acids set forthin any of SEQ ID NOS: 237-240 or a sequence of amino acids that exhibitsat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% or more sequence identity to SEQ ID NO: 237-240 andretains the activity of T cell costimulatory signaling.

In particular embodiments, the CAR further comprises a hinge or spacerregion which connects the extracellular antigen binding domain and thetransmembrane domain. This hinge or spacer region can be used to achievedifferent lengths and flexibility of the resulting CAR. Examples of thehinge or spacer region that can be used include, but are not limited to,Fc fragments of antibodies or fragments or derivatives thereof, hingeregions of antibodies, or fragments or derivatives thereof, C_(H)2regions of antibodies, C_(H)3 regions of antibodies, artificial spacersequences, for example peptide sequences, or combinations thereof. Otherhinge or spacer region will be apparent to those of skill in the art andmay be used. In one embodiment, the hinge is an lgG4 hinge or a CD8Ahinge.

In some embodiments, the spacer and transmembrane domain are the hingeand transmembrane domain derived from CD8, such as having an exemplarysequence set forth in SEQ ID NO: 241-243 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 241-243.

Also provided herein is an isolated nucleic acid construct comprising apolynucleotide encoding a PD-1 binding molecule as provided herein. Alsoprovided herein is an isolated nucleic acid construct comprising apolynucleotide encoding a CAR and encoding the PD-1 binding molecule asprovided herein. In some a first nucleic acid encoding the CAR isseparated from a second nucleic acid encoding the PD-1 binding moleculeby a biscistronic element, such as an IRES or a ribosome skip sequence(e.g. T2A or P2A). In some aspects, the construct is an expressionvector for expression of the PD-1 binding molecule and/or CAR in a cell.The expression vector may be a viral vector. Viral vector technology iswell known in the art and is described, for example, in Sambrook et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 2013). A number of viral based systems have been developed forgene transfer into mammalian cells. For example, retroviruses such as,adenovirus vectors are used. In one embodiment, a lentivirus vector isused.

In a further aspect, also provided is an isolated cell or cellpopulation comprising one or more nucleic acid construct as describedabove. Also provided is an isolated cell or cell population that hasbeen genetically modified to express a PD-1 binding molecule and/or CARprovided herein. Thus, provided herein are genetically engineered cellswhich comprise, such as stably express, a CAR provided herein. In oneembodiment, the cell is selected from the group consisting of a T cell,a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatoryT cell, hematopoietic stem cells and/or pluripotent embryonic/inducedstem cells. In some cases, the cell is a T cell, such as a CD4 and/orCD8 T cell. In some embodiments, the cells are autologous to thesubject. For example, in some embodiments, T cells may be isolated froma patient (also called primary T cells) for engineering, e.g.transfection or transduction, with a CAR nucleic acid construct.

In an exemplary example, primary T-cells can be purified ex vivo (CD4cells or CD8 cells or both) and stimulated with a TCR/CD28 agonists,such as anti-CD3/anti-CD28 coated beads. After a 2 or 3 day activationprocess, a recombinant expression vector encoding the CAR can be stablyintroduced into the primary T cells through standard lentiviral orretroviral transduction protocols or plasmid electroporation strategies.Cells can be monitored for secretion of an PD-1 binding molecule and/orCAR expression by, for example, flow cytometry using anti-epitope tag orantibodies that cross-react with native parental molecule. T-cells thatexpress the CAR can be enriched through sorting with anti-epitope tagantibodies or enriched for high or low expression depending on theapplication.

The PD-1 binding molecule and/or CAR engineered T-cells can be assayedfor appropriate function by a variety of means. In some cases, in vitrocytotoxicity, proliferation, or cytokine assays (e.g., IFN-gammaexpression) can be used to assess the function of engineered T-cells.Exemplary standard endpoints are percent lysis of a tumor line,proliferation of the engineered T-cell, or IFN-gamma protein expressionin culture supernatant. In some cases, the ability to stimulateactivation of T cells upon stimulation of the CAR, e.g. via antigen, canbe assessed, such as by monitoring expression of activation markers suchas CD69, CD44, or CD62L, proliferation and/or cytokine production.

Also provided herein are methods for the prevention and/or treatment ofa disease or condition in a subject, such as a cancer, that includesadministering to a subject engineered cells provided herein. Generally,the subject is in need of treatment for the disease or condition.pharmaceutically active amount of a cell and/or of a pharmaceuticalcomposition of the invention.

IV. Polypeptide Expression and Production

Nucleic acid molecules comprising polynucleotides that encode any of theprovided sdAb and PD-1-binding polypeptides are provided. In someembodiments, the provided nucleic acid sequences and particularly DNAsequences encode fusion proteins as provided herein. In any of theforegoing embodiments, the nucleic acid molecule may also encode aleader sequence that directs secretion of the PD-1-binding polypeptide,which leader sequence is typically cleaved such that it is not presentin the secreted polypeptide. The leader sequence may be a native heavychain (or VHH) leader sequence, or may be another heterologous leadersequence.

Nucleic acid molecules can be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Vectors comprising nucleic acids that encode the PD-1-bindingpolypeptides described herein are provided. Such vectors include, butare not limited to, DNA vectors, phage vectors, viral vectors,retroviral vectors, etc. In some embodiments, a vector is selected thatis optimized for expression of polypeptides in a desired cell type, suchas CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors aredescribed, for example, in Running Deer et al., Biotechnol. Prog.20:880-889 (2004).

In particular, a DNA vector that encodes a desired PD-1-bindingpolypeptides, such as a fusion protein, can be used to facilitate themethods of preparing the PD-1-binding polypeptides described herein andto obtain significant quantities. The DNA sequence can be inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. A variety of host-vector systems may beutilized to express the protein-coding sequence. These include mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Dependingon the host-vector system utilized, any one of a number of suitabletranscription and translation elements may be used.

The disclosure also provides methods of producing a PD-1-bindingpolypeptides by culturing a cell under conditions that lead toexpression of the polypeptide, wherein the cell comprises an isolatednucleic acid molecule encoding a PD-1-binding polypeptide describedherein, and/or vectors that include these isolated nucleic acidsequences.

In some embodiments, a PD-1-binding polypeptide may be expressed inprokaryotic cells, such as bacterial cells; or in eukaryotic cells, suchas fungal cells (such as yeast), plant cells, insect cells, andmammalian cells. Such expression may be carried out, for example,according to procedures known in the art. Exemplary eukaryotic cellsthat may be used to express polypeptides include, but are not limitedto, COS cells, including COS 7 cells; 293 cells, including 293-6E cells;CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells;PER.C6® cells (Crucell); and NSO cells. In some embodiments, thePD-1-binding polypeptides may be expressed in yeast. See, e.g., U.S.Publication No. US 2006/0270045 A1. In some embodiments, a particulareukaryotic host cell is selected based on its ability to make desiredpost-translational modifications to the polypeptide. For example, insome embodiments, CHO cells produce polypeptides that have a higherlevel of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids (such as vectors) into adesired host cell may be accomplished by any method, including but notlimited to, calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection, etc. Nonlimiting exemplary methods aredescribed, for example, in Sambrook et al., Molecular Cloning, ALaboratory Manual, 3^(rd) ed. Cold Spring Harbor Laboratory Press(2001). Nucleic acids may be transiently or stably transfected in thedesired host cells, according to any suitable method.

Host cells comprising any of the nucleic acids or vectors describedherein are also provided. In some embodiments, a host cell thatexpresses a PD-1-binding polypeptide described herein is provided. ThePD-1-binding polypeptides expressed in host cells can be purified by anysuitable method. Such methods include, but are not limited to, the useof affinity matrices or hydrophobic interaction chromatography. Suitableaffinity ligands include the ROR1 ECD and agents that bind Fc regions.For example, a Protein A, Protein G, Protein A/G, or an antibodyaffinity column may be used to bind the Fc region and to purify aPD-1-binding polypeptide that comprises an Fc region. Hydrophobicinteractive chromatography, for example, a butyl or phenyl column, mayalso suitable for purifying some polypeptides such as antibodies. Ionexchange chromatography (for example anion exchange chromatographyand/or cation exchange chromatography) may also suitable for purifyingsome polypeptides such as antibodies. Mixed-mode chromatography (forexample reversed phase/anion exchange, reversed phase/cation exchange,hydrophilic interaction/anion exchange, hydrophilic interaction/cationexchange, etc.) may also suitable for purifying some polypeptides suchas antibodies. Many methods of purifying polypeptides are known in theart.

In some embodiments, the PD-1-binding polypeptide is produced in acell-free system. Nonlimiting exemplary cell-free systems are described,for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009);Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol.Adv. 21: 695-713 (2003).

In some embodiments, PD-1-binding polypeptides prepared by the methodsdescribed above are provided. In some embodiments, the PD-1-bindingpolypeptide is prepared in a host cell. In some embodiments, thePD-1-binding polypeptide is prepared in a cell-free system. In someembodiments, the PD-1-binding polypeptide is purified. In someembodiments, a cell culture media comprising a PD-1-binding polypeptideis provided.

In some embodiments, compositions comprising antibodies prepared by themethods described above are provided. In some embodiments, thecomposition comprises a PD-1-binding polypeptide prepared in a hostcell. In some embodiments, the composition comprises a PD-1-bindingpolypeptide prepared in a cell-free system. In some embodiments, thecomposition comprises a purified PD-1-binding polypeptide.

V. Pharmaceutical Compositions and Formulations

Provided herein are pharmaceutical compositions containing any of thePD-1-binding polypeptides provided herein or engineered cells expressingthe same. In some embodiments, PD-1-binding polypeptides, such as fusionproteins of the disclosure (also referred to herein as “activecompounds”), and derivatives, fragments, analogs and homologs thereof,can be incorporated into pharmaceutical compositions suitable foradministration. In some embodiments, engineered cells expressing achimeric receptor, such as a chimeric antigen receptor, containing aPD-1-binding polypeptide provided herein can be incorporated intopharmaceutical compositions suitable for administration.

Such compositions typically contain a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutically acceptable carrier”is intended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Suitable examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated. Supplementary active compounds canalso be incorporated into the compositions.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intratumoral, oral (e.g., inhalation), transdermal (i.e.,topical), transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid (EDTA); buffers such asacetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscan also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration. Thesepharmaceutical compositions can be included in diagnostic kits withinstructions for use.

Pharmaceutical compositions are administered in an amount effective fortreatment or prophylaxis of the specific indication. The therapeuticallyeffective amount is typically dependent on the weight of the subjectbeing treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In some embodiments, the pharmaceutical composition maybe administered in an amount in the range of about 50 μg/kg body weightto about 50 mg/kg body weight per dose. In some embodiments, thepharmaceutical composition may be administered in an amount in the rangeof about 100 μg/kg body weight to about 50 mg/kg body weight per dose.In some embodiments, the pharmaceutical composition may be administeredin an amount in the range of about 100 μg/kg body weight to about 20mg/kg body weight per dose. In some embodiments, the pharmaceuticalcomposition may be administered in an amount in the range of about 0.5mg/kg body weight to about 20 mg/kg body weight per dose.

In some embodiments, the pharmaceutical composition may be administeredin an amount in the range of about 10 mg to about 1,000 mg per dose. Insome embodiments, the pharmaceutical composition may be administered inan amount in the range of about 20 mg to about 500 mg per dose. In someembodiments, the pharmaceutical composition may be administered in anamount in the range of about 20 mg to about 300 mg per dose. In someembodiments, the pharmaceutical composition may be administered in anamount in the range of about 20 mg to about 200 mg per dose.

The pharmaceutical composition may be administered as needed tosubjects. In some embodiments, an effective dose of the pharmaceuticalcomposition is administered to a subject one or more times. In variousembodiments, an effective dose of the pharmaceutical composition isadministered to the subject once a month, less than once a month, suchas, for example, every two months, every three months, or every sixmonths. In other embodiments, an effective dose of the pharmaceuticalcomposition is administered more than once a month, such as, forexample, every two weeks, every week, twice per week, three times perweek, daily, or multiple times per day. An effective dose of thepharmaceutical composition is administered to the subject at least once.In some embodiments, the effective dose of the pharmaceuticalcomposition may be administered multiple times, including for periods ofat least a month, at least six months, or at least a year. In someembodiments, the pharmaceutical composition is administered to a subjectas-needed to alleviate one or more symptoms of a condition.

VI. Methods of Treatment and Uses

The PD-1-binding polypeptides or engineered cells expressing the samedescribed herein are useful in a variety of therapeutic, diagnostic andprophylactic indications. For example, the PD-1-binding polypeptides orengineered cells are useful in treating a variety of diseases anddisorders in a subject. Such methods and uses include therapeuticmethods and uses, for example, involving administration of the moleculesor engineered cells, or compositions containing the same, to a subjecthaving a disease, condition, or disorder, such as a tumor or cancer. Insome embodiments, the molecule ore engineered cell is administered in aneffective amount to effect treatment of the disease or disorder. Usesinclude uses of molecules containing the PD-1-binding polypeptides orengineered cells in such methods and treatments, and in the preparationof a medicament in order to carry out such therapeutic methods. In someembodiments, the methods are carried out by administering thePD-1-binding polypeptides or engineered cells, or compositionscomprising the same, to the subject having or suspected of having thedisease or condition. In some embodiments, the methods thereby treat thedisease or condition or disorder in the subject.

In one embodiment, a PD-1-binding polypeptide or engineered cell of thedisclosure may be used as therapeutic agents. Such agents will generallybe employed to diagnose, prognose, monitor, treat, alleviate, and/orprevent a disease or pathology in a subject. A therapeutic regimen iscarried out by identifying a subject, e.g., a human patient or othermammal suffering from (or at risk of developing) a disorder usingstandard methods. In some cases, a subject is selected that is known,suspected or that has been identified as having a tumor expressing PD-1.A PD-1-binding polypeptide or engineered cell is administered to thesubject. A PD-1-binding polypeptide or engineered cell is administeredto the subject and will generally have an effect due to its binding withthe target(s).

In some embodiments, a provided PD-1 polypeptide multi-specificpolypeptide construct or engineered cell is capable of modulating, e.g.increasing, an immune response when administered to a subject, such asby engagement of CD3 and/or a CD3 signal in a cell and/or by blockingthe interaction of PD-1 and PD-L1/PD-L2. In some embodiments, providedherein is a method of modulating an immune response in a subject byadministering a therapeutically effective amount of a providedmultispecific construct or engineered cell, or pharmaceuticalcompositions thereof. In some embodiments, the method of modulating animmune response increases or enhances an immune response in a subject.For example, the increase or enhanced response may be an increase incell-mediated immunity. In some examples, the method increases T-cellactivity, such as cytolytic T-cell (CTL) activity. In some embodiments,the modulated (e.g., increased) immune response is against a tumor orcancer.

In some embodiments, administration of a PD-1-binding polypeptide, suchas an PD-1-Fc fusion protein or a multispecific construct containing anFc region, may activate innate immune cells via engagement of FcγRsthrough the Fc-region of the multispecific polypeptide construct.Administration of such multispecific polypeptide constructs may agonize,stimulate, activate, and/or augment innate immune cell effectorfunctions, including ADCC, cytokine release, degranulation and/or ADCP.In the case of a constrained multispecific polypeptide construct,administration of such multispecific polypeptide constructs may activateT-cells once the linker(s) joining the first and second component iscleaved by a protease and/or upon binding of tumor antigen on a targetcell (e.g. tumor cell), thereby allowing the anti-CD3 binding portion tobind CD3ε on the T cells. In some cases, administration of themultispecific polypeptide constructs may agonize, stimulate, activate,and/or augment CD3-mediated T cell activation, cytotoxicity, cytokinerelease and/or proliferation.

In some embodiments, the provided methods are for treating a disease orcondition in a subject by administering a therapeutically effectiveamount of any of the provided PD-1-binding polypeptides or engineeredcells or pharmaceutical compositions thereof. In some embodiments, thedisease or condition is a tumor or a cancer. Generally, alleviation ortreatment of a disease or disorder involves the lessening of one or moresymptoms or medical problems associated with the disease or disorder.For example, in the case of cancer, the therapeutically effective amountof the drug can accomplish one or a combination of the following: reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., todecrease to some extent and/or stop) cancer cell infiltration intoperipheral organs; inhibit tumor metastasis; inhibit, to some extent,tumor growth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. In some embodiments, a composition of thisdisclosure can be used to prevent the onset or reoccurrence of thedisease or disorder in a subject, e.g., a human or other mammal, such asa non-human primate, companion animal (e.g., cat, dog, horse), farmanimal, work animal, or zoo animal. The terms subject and patient areused interchangeably herein.

In some embodiments, the PD-1-binding polypeptides or engineered cells,or pharmaceutical compositions thereof, can be used to inhibit growth ofmammalian cancer cells (such as human cancer cells). A method oftreating cancer can include administering an effective amount of any ofthe pharmaceutical compositions described herein to a subject withcancer. The effective amount of the pharmaceutical composition can beadministered to inhibit, halt, or reverse progression of cancers. Humancancer cells can be treated in vivo, or ex vivo. In ex vivo treatment ofa human patient, tissue or fluids containing cancer cells are treatedoutside the body and then the tissue or fluids are reintroduced backinto the patient. In some embodiments, the cancer is treated in a humanpatient in vivo by administration of the therapeutic composition intothe patient.

Non-liming examples of disease include: all types of cancers (breast,lung, colorectal, prostate, melanomas, head and neck, pancreatic, etc.),rheumatoid arthritis, Crohn's disuse, SLE, cardiovascular damage,ischemia, etc. For example, indications would include leukemias,including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblasticdiseases including multiple myeloma, and solid tumors, including lung,colorectal, prostate, pancreatic, and breast, including triple negativebreast cancer. For example, indications include bone disease ormetastasis in cancer, regardless of primary tumor origin; breast cancer,including by way of non-limiting example, ER/PR+ breast cancer, Her2+breast cancer, triple-negative breast cancer; colorectal cancer;endometrial cancer; gastric cancer; glioblastoma; head and neck cancer,such as esophageal cancer; lung cancer, such as by way of non-limitingexample, non-small cell lung cancer; multiple myeloma ovarian cancer;pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renalcancer, such as by way of nonlimiting example, renal cell carcinoma;and/or skin cancer, such as by way of nonlimiting example, squamous cellcancer, basal cell carcinoma, or melanoma. In some embodiments, thecancer is a squamous cell cancer. In some embodiments, the cancer is askin squamous cell carcinoma. In some embodiments, the cancer is anesophageal squamous cell carcinoma. In some embodiments, the cancer is ahead and neck squamous cell carcinoma. In some embodiments, the canceris a lung squamous cell carcinoma.

In some embodiments, the PD-1-binding polypeptides or engineered cells,or pharmaceutical compositions thereof, or are useful in treating,alleviating a symptom of, ameliorating and/or delaying the progressionof a cancer or other neoplastic condition. In some embodiments, thecancer is bladder cancer, breast cancer, uterine/cervical cancer,ovarian cancer, prostate cancer, testicular cancer, esophageal cancer,gastrointestinal cancer, pancreatic cancer, colorectal cancer, coloncancer, kidney cancer, head and neck cancer, lung cancer, stomachcancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,myeloma, sarcoma, and virus-related cancer. In certain embodiments, thecancer is a metastatic cancer, refractory cancer, or recurrent cancer.

In some embodiments, a therapeutically effective amount of aPD-1-binding polypeptide, such as a fusion protein or multispecificpolypeptide construct, of the disclosure relates generally to the amountneeded to achieve a therapeutic objective. Typically, precise amount ofthe compositions of the present disclosure to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject).

In some embodiments, a therapeutically effective dose may be, by way ofnonlimiting example, from about 0.01 μg/kg body weight to about 10 mg/kgbody weight. In some embodiments, the therapeutically effective dose maybe, by way of nonlimiting example, from about 0.01 mg/kg body weight toabout 5-10 mg/kg body weight. Common dosing frequencies may range, forexample, from twice daily to once a week.

In some embodiments, a therapeutic amount of an engineered cellcomposition of the present disclosure is administered. It can generallybe stated that a pharmaceutical composition comprising engineered cells,e.g., T cells, as described herein may be administered at a dosage of10⁴ to 10⁹ cells/kg body weight, such as 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. Engineeredcell compositions, such as T cell compositions, may also be administeredmultiple times at these dosages. The cells can be administered by usinginfusion techniques that are commonly known in immunotherapy (see, e.g.,Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular disorder. Methods forthe screening of PD-1-binding polypeptides, or engineered cellscontaining the same, that possess the desired specificity include, butare not limited to, enzyme linked immunosorbent assay (ELISA) and otherimmunologically mediated techniques known within the art. A variety ofmeans are known for determining if administration of the providedPD-1-binding polypeptides or engineered cells sufficiently modulatesimmunological activity by eliminating, sequestering, or inactivatingimmune cells mediating or capable of mediating an undesired immuneresponse; inducing, generating, or turning on immune cells that mediateor are capable of mediating a protective immune response; changing thephysical or functional properties of immune cells; or a combination ofthese effects. Examples of measurements of the modulation ofimmunological activity include, but are not limited to, examination ofthe presence or absence of immune cell populations (using flowcytometry, immunohistochemistry, histology, electron microscopy,polymerase chain reaction (PCR)); measurement of the functional capacityof immune cells including ability or resistance to proliferate or dividein response to a signal (such as using T-cell proliferation assays andpepscan analysis based on 3H-thymidine incorporation followingstimulation with anti-CD3 antibody, anti-T-cell receptor antibody,anti-CD28 antibody, calcium ionophores, PMA (phorbol 12-myristate13-acetate) antigen presenting cells loaded with a peptide or proteinantigen; B cell proliferation assays); measurement of the ability tokill or lyse other cells (such as cytotoxic T cell assays); measurementsof the cytokines, chemokines, cell surface molecules, antibodies andother products of the cells (e.g., by flow cytometry, enzyme-linkedimmunosorbent assays, Western blot analysis, protein microarrayanalysis, immunoprecipitation analysis); measurement of biochemicalmarkers of activation of immune cells or signaling pathways withinimmune cells (e.g., Western blot and immunoprecipitation analysis oftyrosine, serine or threonine phosphorylation, polypeptide cleavage, andformation or dissociation of protein complexes; protein array analysis;DNA transcriptional, profiling using DNA arrays or subtractivehybridization); measurements of cell death by apoptosis, necrosis, orother mechanisms (e.g., annexin V staining, TUNEL assays, gelelectrophoresis to measure DNA laddering, histology; fluorogenic caspaseassays, Western blot analysis of caspase substrates); measurement of thegenes, proteins, and other molecules produced by immune cells (e.g.,Northern blot analysis, polymerase chain reaction, DNA microarrays,protein microarrays, 2-dimensional gel electrophoresis, Western blotanalysis, enzyme linked immunosorbent assays, flow cytometry); andmeasurement of clinical symptoms or outcomes such as improvement ofautoimmune, neurodegenerative, and other diseases involvingself-proteins or self-polypeptides (clinical scores, requirements foruse of additional therapies, functional status, imaging studies) forexample, by measuring relapse rate or disease severity.

The provided PD-1-binding polypeptides are also useful in a variety ofdiagnostic and prophylactic formulations. In one embodiment, aPD-1-binding polypeptide is administered to patients that are at risk ofdeveloping one or more of the aforementioned disorders. A patient's ororgan's predisposition to one or more of the disorders can be determinedusing genotypic, serological or biochemical markers.

In another embodiment of the disclosure, a PD-1-binding polypeptide orengineered cell is administered to human individuals diagnosed with aclinical indication associated with one or more of the aforementioneddisorders. Upon diagnosis, such a therapeutic agent is administered tomitigate or reverse the effects of the clinical indication.

Combination Therapy

PD-1-binding polypeptides or engineered cells of the present disclosurecan be administered alone or in combination with other modes oftreatment, such as other anti-cancer agents. They can be providedbefore, substantially contemporaneous with, or after other modes oftreatment (i.e., concurrently or sequentially). In some embodiments, themethod of treatment described herein can further include administering:radiation therapy, chemotherapy, vaccination, targeted tumor therapy,CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokinetherapy, surgical resection, chromatin modification, ablation,cryotherapy, an antisense agent against a tumor target, a siRNA agentagainst a tumor target, a microRNA agent against a tumor target or ananti-cancer/tumor agent, or a biologic, such as an antibody, cytokine,or receptor extracellular domain-Fc fusion.

In some embodiments, a PD-1-binding polypeptide provided herein is givenconcurrently with one or more chemotherapeutic agent, CAR-T (chimericantigen receptor T-cell) therapy, oncolytic virus therapy, cytokinetherapy, and/or agents that target other checkpoint molecules, such asVISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

In some embodiments, the PD-1-binding polypeptide or engineered cells ofthe present disclosure is used in combination with other anti-tumoragents, such as anti-HER-2 antibodies, anti-CD20 antibodies, anepidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosinekinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVA®),platelet derived growth factor inhibitors (e.g., GLEEVEC® (ImatinibMesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, CTLA4inhibitors (e.g., anti-CTLA antibody ipilimumab (YERVOY®)), PD-1inhibitors (e.g., anti-PD1 antibodies, BMS-936558), PDL1 inhibitors(e.g., anti-PDL1 antibodies, MPDL3280A), PDL2 inhibitors (e.g.,anti-PDL2 antibodies), cytokines, antagonists (e.g., neutralizingantibodies) that bind to one or more of the following targets ErbB2,ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, orVEGF receptor(s), TRAIL/Apo2, and other bioactive and organic chemicalagents, etc.

In some embodiments, a PD-1-binding polypeptide or engineered cellprovided herein is given concurrently with a PD-1/PD-L1 therapy.Examples of PD-1/PD-L1 therapy include nivolumab (BMS); pidilizumab(CureTech, CT-011), pembrolizumab (Merck); durvalumab(Medimmune/AstraZeneca); atezolizumab (Genentech/Roche); avelumab(Pfizer); AMP-224 (Amplimmune); BMS-936559; AMP-514 (Amplimmune);MDX-1105 (Merck); TSR-042 (Tesaro/AnaptysBio, ANB-011); STI-A1010(Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and otheragents that are directed against programmed death-1 (PD-1) or programmeddeath ligand 1 (PD-L1).

In some embodiments, the PD-1-binding polypeptide or engineered cell ofthe present disclosure may be used in combination with achemotherapeutic agent. Examples of chemotherapeutic agents include, butare not limited to, alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Further nonlimiting exemplary chemotherapeutic agents includeanti-hormonal agents that act to regulate or inhibit hormone action oncancers such as anti-estrogens and selective estrogen receptormodulators (SERMs), including, for example, tamoxifen (includingNOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® toremifene;aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN®exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA®letrozole, and ARIMIDEX® anastrozole; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in abherant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGFexpression inhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expressioninhibitor; vaccines such as gene therapy vaccines, for example,ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN®(aldesleukin) rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX®GnRH agonist; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In some embodiments, the PD-1-binding polypeptide and the additionalagent are formulated into a single therapeutic composition, and thePD-1-binding polypeptide and additional agent are administeredsimultaneously. Alternatively, the PD-1-binding polypeptide orengineered cell and the additional agent are separate from each other,e.g., each is formulated into a separate therapeutic composition, andthe PD-1-binding polypeptide or engineered cell and the additional agentare administered simultaneously, or the PD-1-binding polypeptide orengineered cell and the additional agent are administered at differenttimes during a treatment regimen. For example, the PD-1-bindingpolypeptide or engineered cell is administered prior to theadministration of the additional agent, the PD-1-binding polypeptide orengineered cell is administered subsequent to the administration of theadditional agent, or the PD-1-binding polypeptide or engineered cell andthe additional agent are administered in an alternating fashion. ThePD-1-binding polypeptide and additional agent may be administered insingle doses or in multiple doses.

In some embodiments, the PD-1-binding polypeptide or engineered cell andthe additional agent(s) are administered simultaneously. For example,the PD-1-binding polypeptide and the additional agent(s) can beformulated in a single composition or administered as two or moreseparate compositions. In some embodiments, the PD-1-binding polypeptideor engineered cell and the additional agent(s) are administeredsequentially, or the PD-1-binding polypeptide or engineered cell and theadditional agent are administered at different times during a treatmentregimen.

VII. Exemplary Embodiments

Among the provided embodiments are:

1. A PD-1-binding polypeptide construct, comprising at least one heavychain only variable domain (PD-1 VHH domain) that specifically bindsPD-1 and one or more additional binding domain that binds to a targetother than PD-1.

2. The PD-1-binding construct of embodiment 1, wherein the at least oneVHH domain comprises a complementarity determining region 1 (CDR1)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 268, 272, 273 and 313; a complementarity determining region 2(CDR2) comprising an amino acid sequence set forth in SEQ ID NO: 278 or314; and a complementarity determining region 3 (CDR3) comprising anamino acid sequence set forth in SEQ ID NO: 283 or 315.

3. A PD-1-binding construct, comprising at least one heavy chain onlyvariable domain (PD-1 VHH domain) comprising a complementaritydetermining region 1 (CDR1) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 268, 272, 273 and 313; acomplementarity determining region 2 (CDR2) comprising an amino acidsequence set forth in SEQ ID NO: 278 or 314; and a complementaritydetermining region 3 (CDR3) comprising an amino acid sequence selectedset forth in SEQ ID NO: 283.

4. The PD-1-binding polypeptide construct of any of embodiments 1-3,wherein the PD-1 is a human PD-1.

5. The PD-1-binding polypeptide construct of any of embodiments 1-4,wherein the at least one PD-1 VHH domain is humanized.

6. The PD-1-binding polypeptide construct of any of embodiments 1, 2, 4and 5, wherein the one or more additional binding domains binds to anactivating receptor on an immune cell.

7. The PD-1-binding polypeptide construct of embodiment 6, wherein theimmune cell is a T cell.

8. The PD-1-binding polypeptide construct of embodiment 6 or embodiment7, wherein the activating receptor is CD3 (CD3ε).

9. The PD-1-binding polypeptide construct of embodiment 8 that isbispecific for PD-1 and CD3.

10. The PD-1-binding polypeptide construct of embodiment 9, wherein theimmune cell is a Natural Killer (NK) cell.

11. The PD-1-binding polypeptide construct of embodiment 6 or embodiment10, wherein the activating receptor is CD16 (CD16a).

12. The PD-1-binding polypeptide construct of embodiment 11 that isbispecific for PD-1 and CD16a.

13. The PD-1-binding polypeptide construct of any of embodiments 1, 2, 4and 5, wherein the one or more additional domains binds to a tumorassociated antigen (TAA).

14. The PD-1-binding polypeptide construct of any of embodiments 1, 2, 4and 5, wherein the one or more additional binding domain binds to acytokine receptor.

15. The PD-1-binding polypeptide construct of any of embodiments 1, 2and 4-14, wherein the one or more additional binding domain comprises anantibody or antigen-binding fragment thereof.

16. The PD-1-binding polypeptide construct of any of embodiments 1, 2and 4-15, wherein the one or more additional binding domain ismonovalent.

17. The PD-1-binding polypeptide construct of embodiment 16, wherein theantibody or antigen-binding fragment thereof is an Fv, adisulfide-stabilized Fv (dsFv), scFv, a Fab, a single domain antibody(sdAb), a VNAR, or a VHH.

18. The PD-1-binding polypeptide construct of embodiment 14, wherein theone or more additional binding domain is a cytokine or is a truncatedfragment or variant thereof capable of binding to the cytokine receptor.

19. The PD-1-binding polypeptide construct of embodiment 18, wherein thecytokine is an interferon that is a type I interferon or a type IIinterferon, is a truncated fragment or variant of a type I interferon oris a truncated fragment or variant of a type II interferon.

20. The PD-1-binding polypeptide construct of embodiment 19, wherein:

the type I interferon is an IFN-alpha or an IFN-beta or is a truncatedfragment or variant thereof; or

the type II interferon is an IFN-gamma or is a truncated fragment orvariant thereof.

21. The PD-1-binding polypeptide construct of any of embodiments 1-20,wherein the polypeptide comprises an immunoglobulin Fc region.

22. The PD-1-binding polypeptide construct of any of embodiments 1, 2and 4-21, wherein the polypeptide comprises an immunoglobulin Fc regionthat links the at least one single domain antibody and the one or moreadditional binding domain.

23. The PD-1-binding polypeptide construct of any of embodiments 1-22that is a dimer.

24. The PD-1-binding polypeptide construct of any of embodiments 21-23,wherein the Fc region is a homodimeric Fc region.

25. The PD-1-binding polypeptide construct of any of embodiments 21-24,wherein the Fc region comprises the sequence of amino acids set forth inany of SEQ ID NOS: 8, 10, 11, 12 or 13, or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 8,10, 11, 12 or 13.

26. The PD-1-binding polypeptide construct of any of embodiments 21-24,wherein the Fc region is a human IgG1.

27. The PD-1-binding polypeptide construct of embodiment 26, wherein theFc region comprises the sequence of amino acids set forth in SEQ ID NO:8or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 8.

28. The PD-1-binding polypeptide construct of any of embodiments 21-23,wherein the Fc region is a heterodimeric Fc region.

29. The PD-1-binding polypeptide construct of any of embodiments 21-28,wherein the Fc region exhibits effector function.

30. The PD-1-binding polypeptide construct of any of embodiments 21-29,wherein the Fc region comprises a polypeptide comprising one or moreamino acid modification that reduces effector function and/or reducesbinding to an effector molecule selected from an Fc gamma receptor orC1q.

31. The PD-1-binding polypeptide construct of embodiment 30, wherein theone or more amino acid modification is deletion of one or more ofGlu233, Leu234 or Leu235.

32. The PD-1-binding polypeptide construct of embodiment 30 orembodiment 31, wherein the Fc region comprises the sequence of aminoacids set forth in SEQ ID NO:9 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9.

33. The PD-1-binding polypeptide construct of any of embodiments 1-32,wherein the at least one PD-1 VHH domain comprises the sequence setforth in any of SEQ ID NOS: 251-267 or 284, or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS:251-267 or 284 and binds PD-1.

34. The PD-1-binding polypeptide construct of any of embodiments 1-33,wherein the at least one PD-1 VHH domain comprises the sequence setforth in (i) SEQ ID NO:284, (ii) a humanized variant of SEQ ID NO:284,or (iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:284 and binds PD-1.

35. The PD-1-binding polypeptide construct of any of embodiments 1-34,wherein the at least one PD-1 VHH domain comprises the sequence setforth in SEQ ID NO:284.

36. The PD-1-binding polypeptide of any of embodiments 1-34, wherein theat least one PD-1 VHH domain comprises a CDR1 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 268, 272 and273; a CDR2 comprising an amino acid sequence set forth in SEQ ID NO:278; and a CDR3 comprising an amino acid sequence set forth in SEQ IDNO: 283.

37. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36, wherein the at least one PD-1 VHH domain comprises a CDR1, CDR2and CDR3 set forth in SEQ ID NOS: 268, 278, and 283, respectively; SEQID NOS: 272, 278, and 283, respectively; or SEQ ID NOS: 273, 278, and283, respectively.

38. The PD-1-binding polypeptide construct of any of embodiments 1-34,36 and 37, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in any one of SEQ ID NOs: 251-267 or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toSEQ ID NO: 251-267 and binds PD-1.

39. The PD-1-binding polypeptide construct of embodiments 1-34 and36-38, wherein the at least one PD-1 VHH domain comprises the sequenceof amino acids set forth in any one of SEQ ID NOS: 251-267.

40. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 257 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 257 and binds PD-1.

41. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-40, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 257.

42. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 251 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 251 and binds PD-1.

43. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 252 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 252 and binds PD-1.

44. The PD-1 binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 253 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 253 and binds PD-1.

45. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 254 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 254 and binds PD-1.

46. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 255 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 255 and binds PD-1.

47. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 256 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 256 and binds PD-1.

48. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 258 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 258 and binds PD-1.

49. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 259 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 259 and binds PD-1.

50. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 260 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 260 and binds PD-1.

51. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 261 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 261 and binds PD-1.

52. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 262 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 262 and binds PD-1.

53. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 263 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 263 and binds PD-1.

54. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 264 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 264 and binds PD-1.

55. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 265 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 265 and binds PD-1.

56. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 266 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 266 and binds PD-1.

57. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises thesequence of amino acids set forth in SEQ ID NO: 267 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 267 and binds PD-1.

58. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain comprises least 95%sequence identity to SEQ ID NO: 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267 or 284 and binds PD-1.

59. The PD-1-binding polypeptide construct of any of embodiments 1-34and 36-39, wherein the at least one PD-1 VHH domain is set forth in SEQID NO: 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267 or 284.

60. A multispecific polypeptide construct, comprising: (a) a firstcomponent comprising a heterodimeric Fc region comprising a first Fcpolypeptide and a second Fc polypeptide and (b) a second componentcomprising an anti-CD3 antibody or antigen-binding fragment comprising avariable heavy chain region (VH) and a variable light chain region (VL),wherein:

the VH and VL that comprise the anti-CD3 antibody or antigen bindingfragment are linked to opposite polypeptides of the heterodimeric Fc;

the first and second components are coupled by a linker, wherein theheterodimeric Fc region is positioned N-terminal to the anti-CD3antibody;

one or both of the first and second components comprises at least oneantigen binding domain that binds a tumor associated antigen (TAA); and

one or both of the first and second components comprises at least oneheavy chain only variable domain (PD-1 VHH domain).

61. The multispecific polypeptide construct of embodiment 60, whereinthe multispecific polypeptide construct comprises at least (i) a firstpolypeptide comprising the first Fc polypeptide of the heterodimeric Fcregion, the linker and the VH or VL domain of the anti-CD3 antibody orantigen binding fragment; and (ii) a second polypeptide comprising thesecond Fc polypeptide of the heterodimeric Fc region, the linker,optionally the same linker as present in the first polypeptide, and theother of the VH or VL domain of the anti-CD3 antibody or antigen bindingfragment,

wherein one or both of the first and second polypeptide independentlycomprise the at least one antigen binding domain that binds a TAA andthe at least one PD-1 VHH domain.

62. The multispecific polypeptide construct of embodiment 60 orembodiment 61, wherein one or both of the first and second Fcpolypeptides of the heterodimeric Fc region comprises at least onemodification to induce heterodimerization compared to a polypeptide of ahomodimeric Fc region, optionally compared to the Fc polypeptide setforth in SEQ ID NO: 245 or an immunologically active fragment thereof.

63. The multispecific polypeptide construct of embodiment 62, whereineach of the first and second Fc polypeptides of the heterodimeric Fcindependently comprise at least one amino acid modification.

64. The multispecific polypeptide construct of embodiment 63, whereineach of the first and second Fc polypeptides of the heterodimeric Fccomprise a knob-into-hole modification or comprise a charge mutation toincrease electrostatic complementarity of the polypeptides.

65. The multispecific polypeptide construct of embodiment 64, whereinthe amino acid modification is a knob-into-hole modification.

66. The multispecific polypeptide construct of any of embodiments 60-65,wherein the first Fc polypeptide of the heterodimeric Fc comprises themodification selected from among Thr366Ser, Leu368Ala, Tyr407Val, andcombinations thereof and the second Fc polypeptide of the heterodimericFc comprises the modification Thr366Trp.

67. The multispecific polypeptide of embodiment 66, wherein the firstand second Fc polypeptides further comprises a modification of anon-cysteine residue to a cysteine residue, wherein the modification ofthe first polypeptide is at one of the position Ser354 and Tyr349 andthe modification of the second Fc polypeptide is at the other of theposition Ser354 and Tyr349.

68. The multispecific polypeptide construct of any of embodiments 60-64,wherein the amino acid modification is a charge mutation to increaseelectrostatic complementarity of the polypeptides.

69. The multispecific polypeptide construct of any of embodiments 60-64and 68, wherein the first and/or second Fc polypeptides or each of thefirst and second Fc polypeptide comprise a modification in complementarypositions, wherein the modification is replacement with an amino acidhaving an opposite charge to the complementary amino acid of the otherpolypeptide.

70. The multispecific polypeptide construct of any of embodiments 60-69,wherein one of the first or second Fc polypeptide of the heterodimericFc further comprises a modification at residue Ile253.

71. The multispecific polypeptide construct of embodiment 70, whereinthe modification is Ile253Arg.

72. The multispecific polypeptide construct of any of embodiments 60-71,wherein one of the first or second Fc polypeptide of the heterodimericFc further comprises a modification at residue His435.

73. The multispecific polypeptide construct of embodiment 72, whereinthe modification is His435Arg.

74. The multispecific polypeptide construct of any of embodiments 60-73,wherein the Fc region comprises a polypeptide that lacks Lys447.

75. The multispecific polypeptide construct of any of embodiments 60-74,wherein the Fc region comprises a polypeptide comprising at least onemodification to enhance FcRn binding.

76. The multispecific polypeptide construct of embodiment 75, whereinthe modification is at a position selected from the group consisting ofMet252, Ser254, Thr256, Met428, Asn434, and combinations thereof.

77. The multispecific polypeptide construct of embodiment 76, whereinthe modification is at a position selected from the group consisting ofMet252Y, Ser254T, Thr256E, Met428L, Met428V, Asn434S, and combinationsthereof.

78. The multispecific polypeptide construct of embodiment 76, whereinthe modification is at position Met252 and at position Met428.

79. The multispecific polypeptide construct of embodiment 78, whereinthe modification is Met252Y and Met428L.

80. The multispecific polypeptide construct of embodiment 76, whereinthe modification is Met252Y and Met428V.

81. The multispecific polypeptide construct of any of embodiments 60-80,wherein the first polypeptide of the heterodimeric Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS:103, 107, 115 or117, and the second polypeptide of the heterodimeric Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS:104, 108, 111,113, 119 or 121.

82. The multispecific polypeptide construct of any of embodiments 1-81,wherein the Fc region comprises a polypeptide comprising at least oneamino acid modification that reduces effector function and/or reducesbinding to an effector molecule selected from an Fc gamma receptor orC1q.

83. The multispecific polypeptide construct of embodiment 82, whereinthe one or more amino acid modification is deletion of one or more ofGlu233, Leu234 or Leu235.

84. The multispecific polypeptide construct of any of embodiments 60-83,wherein the first polypeptide of the heterodimeric Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS: 105, 109, 116 or118 and the second polypeptide of the heterodimeric Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS: 106, 110, 112,114, 120 or 122.

85. The multispecific polypeptide construct of any of embodiment 60-84,wherein the anti-CD3 antibody or antigen binding fragment is monovalent.

86. The multispecific polypeptide construct of any of embodiments 60-85,wherein the anti-CD3 antibody or antigen binding fragment is not asingle chain antibody, optionally is not a single chain variablefragment (scFv).

87. The multispecific polypeptide construct of any of embodiments 60-86,wherein the anti-CD3 antibody or antigen binding fragment is an Fvantibody fragment.

88. The multispecific polypeptide construct of embodiment 87, whereinthe Fv antibody fragment comprises a disulfide stabilized anti-CD3binding Fv fragment (dsFv).

89. The multispecific polypeptide construct of 60-88, wherein theanti-CD3 antibody or antigen-binding fragment comprises a VH CDR1comprising the amino acid sequence TYAMN (SEQ ID NO: 29); a VH CD2comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 30);a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO:31), a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ IDNO: 32); a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ IDNO: 33); and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQID NO: 34).

90. The multispecific polypeptide construct of any of embodiments 60-89,wherein the anti-CD3 antibody or antigen-binding fragment comprises:

a VH having the amino acid sequence of any of SEQ ID NOS: 35-65 or asequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to any of SEQ ID NOS: 35-65; and

a VL having the amino acid sequence of any of SEQ ID NOS: 66-85 and 293or a sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to any of SEQ ID NOS: 66-85 and 293.

91. The multispecific polypeptide construct of any of embodiments 60-90,wherein the anti-CD3 antibody or antigen-binding fragment comprises theamino acid sequence of SEQ ID NO: 47 and the amino acid sequence of SEQID NO: 75.

92. The multispecific polypeptide construct of any of embodiments 60-90,wherein the anti-CD3 antibody or antigen-binding fragment comprises theamino acid sequence of SEQ ID NO: 47 and the amino acid sequence of SEQID NO: 285.

93. The multispecific polypeptide construct of any of embodiment 60-92,wherein the at least one PD-1 VHH domain is positioned amino-terminallyrelative to the Fc region and/or carboxy-terminally relative to the CD3binding region of the multispecific polypeptide construct.

94. The multispecific polypeptide construct of any of embodiments 60-93,wherein

the first polypeptide comprises in order of N-terminus to C-terminus afirst antigen binding domain that binds a TAA, a first polypeptidecomprising the first Fc polypeptide of the heterodimeric Fc region, thelinker, the VH or VL domain of the anti-CD3 antibody or antigen bindingfragment and a second antigen binding domain that binds a TAA; and

the second polypeptide comprises the at least one PD-1 VHH domain andcomprises in order of N-terminus to C-terminus the second Fc polypeptideof the heterodimeric Fc region, the linker, optionally the same linkeras present in the first polypeptide, and the other of the VH or VLdomain of the anti-CD3 antibody or antigen binding fragment, wherein theat least one PD-1 VHH domain is positioned amino-terminally relative tothe Fc region and/or carboxy-terminally relative to the CD3 bindingregion.

95. The multispecific polypeptide construct of any of embodiments 60-94,wherein the multispecific polypeptide construct comprises only one PD-1VHH domain that specifically binds PD-1.

96. The multispecific polypeptide construct of any of embodiments 60-95,wherein the PD-1 VHH domain is positioned amino-terminally relative tothe Fc region of the multispecific construct.

97. The multispecific polypeptide construct of any of embodiments 60-96,wherein

the first polypeptide comprises in order of N-terminus to C-terminus afirst antigen binding domain that binds a TAA, a first polypeptidecomprising the first Fc polypeptide of the heterodimeric Fc region, thelinker, the VH or VL domain of the anti-CD3 antibody or antigen bindingfragment and a second antigen binding domain that binds a TAA; and

the second polypeptide comprises in order of N-terminus to C-terminusthe second Fc polypeptide of the heterodimeric Fc region, the linker,optionally the same linker as present in the first polypeptide, theother of the VH or VL domain of the anti-CD3 antibody or antigen bindingfragment and the PD-1 VHH domain.

98. The multispecific polypeptide construct of any of embodiments 60-95,wherein the PD-1 VHH domain is positioned amino-terminally relative tothe Fc region of the multispecific construct.

99. The multispecific polypeptide construct of any of embodiments 60-95and 98, wherein

the first polypeptide comprises in order of N-terminus to C-terminus afirst antigen binding domain that binds a TAA, a first polypeptidecomprising the first Fc polypeptide of the heterodimeric Fc region, thelinker, the VH or VL domain of the anti-CD3 antibody or antigen bindingfragment and a second antigen binding domain that binds a TAA; and

the second polypeptide comprises in order of N-terminus to C-terminusthe PD-1 VHH domain, the second Fc polypeptide of the heterodimeric Fcregion, the linker, optionally the same linker as present in the firstpolypeptide, and the other of the VH or VL domain of the anti-CD3antibody or antigen binding fragment.

100. The multispecific polypeptide construct of any of embodiments60-99, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in any of SEQ ID NOS: 251-267 or 284, or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ IDNOS: 251-267 or 284 and binds PD-1.

101. The multispecific polypeptide construct of any of embodiments60-100, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:284, (ii) a humanized variant of SEQ IDNO:284, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:284 and binds PD-1.

102. The multispecific polypeptide construct of any of embodiments60-101, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in SEQ ID NO:284.

103. The multispecific polypeptide construct of any of embodiments60-101, wherein the at least one PD-1 VHH domain comprises a CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 268, 272 and 273; a CDR2 comprising an amino acid sequenceset forth in SEQ ID NO: 278; and a CDR3 comprising an amino acidsequence set forth in SEQ ID NO: 283.

104. The multispecific polypeptide construct of any of embodiments60-100 and 103, wherein the at least one PD-1 VHH domain comprises aCDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 268, 278, and 283,respectively; SEQ ID NOS: 272, 278, and 283, respectively; or SEQ IDNOS: 273, 278, and 283, respectively.

105. The multispecific polypeptide construct of any of embodiments60-100, 103 and 104, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in any one of SEQ ID NOs: 251-267or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 251-267 and binds PD-1.

106. The multispecific polypeptide construct of embodiments 60-100 and103-105, wherein the at least one PD-1 VHH domain comprises the sequenceof amino acids set forth in any one of SEQ ID NOS: 251-267.

107. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 257 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 257 and binds PD-1.

108. The multispecific polypeptide construct of any of embodiments60-100 and 103-107, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 257.

109. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 251 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 251 and binds PD-1.

110. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 252 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 252 and binds PD-1.

111. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 253 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 253 and binds PD-1.

112. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 254 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 254 and binds PD-1.

113. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 255 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 255 and binds PD-1.

114. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 256 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 256 and binds PD-1.

115. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 258 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 258 and binds PD-1.

116. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 259 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 259 and binds PD-1.

117. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 260 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 260 and binds PD-1.

118. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 261 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 261 and binds PD-1.

119. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 262 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 262 and binds PD-1.

120. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 263 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 263 and binds PD-1.

121. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 264 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 264 and binds PD-1.

122. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 265 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 265 and binds PD-1.

123. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 266 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 266 and binds PD-1.

124. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprisesthe sequence of amino acids set forth in SEQ ID NO: 267 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 267 and binds PD-1.

125. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain comprises asequence of amino acids that exhibits at least 95% sequence identity toSEQ ID NO: 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267 or 284 and binds PD-1.

126. The multispecific polypeptide construct of any of embodiments60-100 and 103-106, wherein the at least one PD-1 VHH domain is setforth in SEQ ID NO: 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267 or 284.

127. The multispecific polypeptide construct of any of embodiments60-126, wherein one or both of the first and second components comprisesat least one co-stimulatory receptor binding region (CRBR) that binds aco-stimulatory receptor.

128. The multispecific polypeptide construct of embodiment 127, whereinthe at least one co-stimulatory receptor binding region (CRBR) ispositioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct.

129. The multispecific polypeptide construct of embodiment 127 orembodiment 128, wherein the multispecific polypeptide constructcomprises only one co-stimulatory receptor binding region (CRBR).

130. The multispecific polypeptide construct of any of embodiments127-129, wherein the multispecific polypeptide construct comprises twoco-stimulatory receptor binding region (CRBR), optionally which are thesame or different.

131. The multispecific polypeptide construct of any of embodiments127-129, wherein the at least one co-stimulatory receptor binding region(CRBR) is or comprises the extracellular domain or binding fragmentthereof of the native cognate binding partner of the co-stimulatoryreceptor, or a variant thereof that exhibits binding activity to theco-stimulatory receptor.

132. The multispecific polypeptide construct of any of embodiments127-130, wherein the at least one co-stimulatory receptor binding region(CRBR) is an antibody or antigen-binding fragment thereof selected fromthe group consisting of a Fab fragment, a F(ab′)2 fragment, an Fvfragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody,and a single domain light chain antibody.

133. The multispecific polypeptide construct of embodiment 132, whereinthe antibody or antigen-binding fragment thereof is a Fv, a scFv, a Fab,a single domain antibody (sdAb), a VNAR, or a VHH.

134. The multispecific polypeptide construct of embodiment 1132 orembodiment 133, wherein the antibody or antigen-binding fragment is ansdAb.

135. The multispecific polypeptide construct of embodiment 134, whereinthe sdAb is a human or humanized sdAb.

136. The multispecific polypeptide construct of any of embodiments127-135, wherein the at least one co-stimulatory receptor binding region(CRBR) binds a co-stimulatory receptor selected from among 41BB (CD137),OX40 (CD134), CD27, glucocorticoid-induced TNFR-related protein (GITR),CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cellmaturation antigen (BCMA), Transmembrane activator and CAML interactor(TACI), and NKG2D.

137. The multispecific polypeptide construct of any of embodiments127-136, wherein the at least one co-stimulatory receptor binding region(CRBR) binds a co-stimulatory receptor selected from among 41BB (CD137),OX40 (CD134), and glucocorticoid-induced TNFR-related protein (GITR).

138. The multispecific polypeptide construct of any of embodiments127-137, wherein the at least one co-stimulatory receptor binding region(CRBR) comprises the sequence of amino acids set forth in SEQ ID NO:210or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequenceset forth in SEQ ID NO:210 and binds 4-1BB.

139. The multispecific polypeptide construct of any of embodiments60-138, wherein the multispecific polypeptide construct comprises afirst and a second antigen binding domains that binds to a TAA.

140. The multispecific polypeptide construct of embodiment 139, whereinthe antigen binding domains bind to the same tumor-associated antigen(TAA).

141. The multispecific polypeptide construct of embodiment 139 orembodiment 149, wherein the first antigen binding domain and the secondantigen binding domain binds a different or nonoverlapping epitope ofthe same TAA and/or compete for binding to the same TAA.

142. The multispecific polypeptide construct of embodiment 139, whereinthe first antigen binding domain and the second antigen binding domainbind a different TAA.

143. The multispecific polypeptide construct of any of embodiments139-142, wherein one antigen binding domain is positionedamino-terminally relative to the Fc region and one antigen bindingdomain is positioned carboxy-terminally relative to the CD3 bindingregion.

144. The multispecific polypeptide construct of any of embodiments60-143, wherein the antigen binding domain that binds a TAA, orindependently each of the antigen binding domains that binds a TAA,comprises an extracellular domain or binding fragment thereof of thenative cognate binding partner of the TAA, or a variant thereof thatexhibits binding activity to the TAA.

145. The multispecific polypeptide construct of any of embodiments60-143, wherein the antigen binding domain that binds a TAA, orindependently each of the antigen binding domains that binds a TAA, isan antibody or antigen-binding fragment thereof.

146. The multispecific polypeptide construct of embodiment 145, whereinthe antibody or antigen-binding fragment thereof is a Fv, a scFv, a Fab,a single domain antibody (sdAb), a VNAR, or a VHH.

147. The multispecific polypeptide construct of embodiment 145 orembodiment 146, wherein the antibody or antigen-binding fragment is ansdAb.

148. The multispecific polypeptide construct of embodiment 147, whereinthe sdAb is a human or humanized sdAb.

149. The multispecific polypeptide construct of any of embodiments60-148, wherein the antigen binding domain, or independently each of theantigen binding domains, binds to a tumor antigen selected from among1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor,APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9,(Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20,CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44,CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81,CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138,CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10,CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM,EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2,FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRα), GAL3ST1, G-CSF,G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors,Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM,Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor(FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R,IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R,Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin,MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, NotchReceptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta,TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha,TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor,TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

150. The multispecific polypeptide construct of any of embodiments139-149, wherein:

the first polypeptide comprises in order of N-terminus to C-terminus afirst antigen binding domain that binds a TAA, a first polypeptidecomprising the first Fc polypeptide of the heterodimeric Fc region, thelinker, the VH or VL domain of the anti-CD3 antibody or antigen bindingfragment and a second antigen binding domain that binds a TAA; and

the second polypeptide comprises in order of N-terminus to C-terminusone of the PD-1 VHH domain or the CRBR, the second Fc polypeptide of theheterodimeric Fc region, the linker, optionally the same linker aspresent in the first polypeptide, the other of the VH or VL domain ofthe anti-CD3 antibody or antigen binding fragment, and the other of thePD-1 VHH domain or the CRBR.

151. The multispecific polypeptide construct of any of embodiments60-150, wherein the linker is a peptide or polypeptide linker,optionally wherein the linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acids in length.

152. The multispecific polypeptide construct of any of embodiments60-151, wherein the linker is a non-cleavable linker.

153. The multispecific polypeptide construct of embodiment 152, whereinthe non-cleavable linker comprises GS, GGS, GGGGS (SEQ ID NO:125),GGGGGS (SEQ ID NO:126) and combinations thereof.

154. The multispecific polypeptide construct of any of embodiments60-153, wherein the linker is or comprises the sequenceGGGGGSGGGGGSGGGGGS (SEQ ID NO:127).

155. The multispecific polypeptide construct of any of embodiments60-151, wherein the linker is a cleavable linker.

156. The multispecific polypeptide construct of embodiment 155, whereinthe cleavable linker is a polypeptide that functions as a substrate fora protease.

157. The multispecific polypeptide construct of embodiment 156, whereinthe protease is produced by an immune effector cell, by a tumor, or bycells present in the tumor microenvironment.

158. The multispecific polypeptide construct of embodiment 156 orembodiment 157, wherein the protease is produced by an immune effectorcell and the immune effector cell is an activated T cell, a naturalkiller (NK) cell, or an NK T cell.

159. The multispecific polypeptide construct of any of embodiments156-158, wherein the protease is selected from among matriptase, amatrix metalloprotease (MMP), granzyme B, and combinations thereof.

160. The multispecific polypeptide construct of embodiment 159, whereinthe protease is granzyme B.

161. The multispecific polypeptide construct of any of embodiments156-160, wherein the cleavable linker comprises the amino acid sequenceGGSGGGGIEPDIGGSGGS (SEQ ID NO:171).

162. An isolated single domain antibody that binds PD-1, comprising acomplementarity determining region 1 (CDR1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 268, 272 and273; a complementarity determining region 2 (CDR2) comprising an aminoacid sequence set forth in SEQ ID NO: 278; and a complementaritydetermining region 3 (CDR3) comprising an amino acid sequence set forthin SEQ ID NO: 283.

163. The isolated single domain antibody of embodiment 162, comprisingthe amino acid sequence set forth in any of SEQ ID NOS: 251-267 or 284,or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any of SEQ ID NOS: 251-267 or 284 and binds PD-1.

164. The isolated single domain antibody of embodiment 162 or embodiment163, wherein the single domain antibody comprises the sequence set forthin (i) SEQ ID NO:284, (ii) a humanized variant of SEQ ID NO:284, or(iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:284 and binds PD-1.

165. The isolated single domain antibody of any of embodiments 162-164,wherein the sdAb comprises the sequence set forth in SEQ ID NO:284.

166. The isolated single domain antibody of any of embodiments 162-164,wherein the sdAb comprises a CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 268, 272 and 273; aCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 278; anda CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 283.

167. The isolated single domain antibody of any of embodiments 162-164and 166, wherein the sdAb comprises a CDR1, CDR2 and CDR3 set forth inSEQ ID NOS: 268, 278, and 283, respectively; SEQ ID NOS: 272, 278, and283, respectively; or SEQ ID NOS: 273, 278, and 283, respectively.

168. The isolated single domain antibody of any of embodiments 162-164,166 and 167, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in any one of SEQ ID NOs: 251-267 or a sequenceof amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ IDNO: 251-267 and binds PD-1.

169. The isolated single domain antibody of any of embodiments 162-164,and 166-168, wherein the sdAb comprises the sequence of amino acids setforth in any one of SEQ ID NOS: 251-267.

170. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 257 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 257 and bindsPD-1.

171. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 257.

172. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 251 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 251 and bindsPD-1.

173. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 252 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 252 and bindsPD-1.

174. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 253 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 253 and bindsPD-1.

175. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 254 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 254 and bindsPD-1.

176. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 255 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 255 and bindsPD-1.

177. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 256 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 256 and bindsPD-1.

178. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 258 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 258 and bindsPD-1.

179. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 259 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 259 and bindsPD-1.

180. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 260 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 260 and bindsPD-1.

181. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 261 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 261 and bindsPD-1.

182. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 262 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 262 and bindsPD-1.

183. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 263 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 263 and bindsPD-1.

184. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 264 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 264 and bindsPD-1.

185. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 265 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 265 and bindsPD-1.

186. The isolated single domain antibody of embodiment any ofembodiments 162-164 and 166-169, wherein the at least one PD-1 sdAbcomprises the sequence of amino acids set forth in SEQ ID NO: 266 or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toSEQ ID NO: 266 and binds PD-1.

187. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the at least one PD-1 sdAb comprises the sequenceof amino acids set forth in SEQ ID NO: 267 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 267 and bindsPD-1.

188. The isolated single domain antibody of any of embodiments 162-164and 166-169, wherein the sdAb comprises the sequence of amino acids setforth in SEQ ID NO: 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267 or 284.

189. A polynucleotide(s) encoding the PD-1-binding polypeptide of any ofembodiments 1-59.

190. A polynucleotide(s) encoding the multispecific polypeptideconstruct of any of embodiments 60-161.

191. A polynucleotide, comprising a first nucleic acid sequence encodinga first polypeptide of a multispecific construct of any of embodiments60-161 and a second nucleic acid sequence encoding a second polypeptideof the multispecific construct, wherein the first and second nucleicacid sequence are separated by an internal ribosome entry site (IRES),or a nucleic acid encoding a self-cleaving peptide or a peptide thatcauses ribosome skipping.

192. The polynucleotide of embodiment 191, wherein the first nucleicacid sequence and second nucleic acid sequence are operably linked tothe same promoter.

193. The polynucleotide of embodiment 192, wherein the nucleic acidencoding a self-cleaving peptide or a peptide that causes ribosomeskipping is selected from a T2A, a P2A, a E2A or a F2A.

194. A polynucleotide encoding the single domain antibody of any ofembodiments 162-188.

195. A vector, comprising the polynucleotide of any of embodiments189-194.

196. The vector of embodiment 195 that is an expression vector.

197. The vector of embodiment 195 or embodiment 196 that is a viralvector or a eukaryotic vector, optionally wherein the eukaryotic vectoris a mammalian vector.

198. A cell, comprising polynucleotide or polynucleotides of any ofembodiments 189-194, or a vector or vectors of any of embodiments195-197.

199. The cell of embodiment 198, wherein the cell is recombinant orisolated.

200. The cell of embodiment 199, wherein the cell is a mammalian cell.

201. A method of producing a polypeptide, the method comprisingintroducing into a cell a polynucleotide or polynucleotides of any ofembodiments 189-194 or a vector or vectors of any of embodiments 195-197and culturing the cell under conditions to produce the multispecificpolypeptide construct.

202. The method of embodiment 201, further comprising isolating orpurifying the polypeptide from the cell.

203. A polypeptide produced by the method of embodiment 201 orembodiment 202.

204. An engineered immune cell, comprising a binding molecule comprisingthe binding molecule of any of embodiments 1-59 or the single domainantibody of any of embodiments 162-188, optionally wherein the bindingmolecule is secretable from the cell.

205. The engineered cell of embodiment 204, further comprising achimeric antigen receptor (CAR).

206. The engineered immune cell of embodiment 204 or embodiment 205,wherein the cell is a lymphocyte.

207. The engineered immune cell of any of embodiments 205-206, whereinthe cell is a T cell or a natural killer (NK) cell.

208. The engineered immune cell of any of embodiments 205-207, whereinthe CAR comprises an extracellular domain comprising an antigen bindingdomain that binds a TAA, a transmembrane domain; and an intracellularsignaling domain.

209. The engineered immune cell of embodiment 208, wherein theintracellular signaling domain comprises an immunoreceptortyrosine-based activation motif (ITAM) signaling domain, optionallywherein the intracellular signaling domain is or comprises a CD3zetasignaling domain, optionally a human CD3zeta signaling domain.

210. The engineered immune cell of embodiment 209, wherein theintracellular signaling domain further comprises a signaling domain of acostimulatory molecule.

211. A pharmaceutical composition comprising the PD-1-bindingpolypeptide of any of embodiments 1-59, the multispecific polypeptideconstruct of any of embodiments 60-161, the single domain antibody ofany of embodiments 162-188 or the engineered immune cell of any ofembodiments 204-210.

212. The pharmaceutical composition of embodiment 211, comprising apharmaceutically acceptable carrier.

213. The pharmaceutical composition of embodiment 211 or embodiment 212that is sterile.

214. A method of stimulating or inducing an immune response in asubject, the method comprising administering, to a subject in needthereof, the PD-1-binding polypeptide of any of embodiments 1-59, themultispecific polypeptide construct of any of embodiments 60-161, thesingle domain antibody of any of embodiments 162-188 or the engineeredimmune cell of any of embodiments 204-210 or a pharmaceuticalcomposition of embodiment 211-213.

215. The method of embodiment 214, wherein the immune response isincreased against a tumor or cancer, optionally a tumor or a cancer thatexpresses PD-1.

216. The method of embodiment 214 or embodiment 215, wherein the methodtreats a disease or condition in the subject.

217. A method of treating a disease or condition in a subject, themethod comprising administering, to a subject in need thereof, atherapeutically effective amount of the PD-1-binding polypeptide of anyof embodiments 1-59, the multispecific polypeptide construct of any ofembodiments 60-161, the single domain antibody of any of embodiments162-188 or the engineered immune cell of any of embodiments 204-210 or apharmaceutical composition of embodiment 211-213.

218. The method of embodiment 216 or embodiment 217, wherein the diseaseor condition is a tumor or a cancer.

219. The method of any of embodiments 214-218, wherein said subject is ahuman.

220. The PD-1-binding construct of embodiment 1, wherein the at leastone VHH domain comprises a complementarity determining region 1 (CDR1)comprising an amino acid sequence set forth in SEQ ID NO:300;complementarity determining region 2 (CDR2) comprising an amino acidsequence set forth in SEQ ID NO: 301; and a complementarity determiningregion 3 (CDR3) comprising an amino acid sequence set forth in SEQ IDNO: 302.

221. The PD-1-binding construct of embodiment 1, wherein the at leastone VHH domain comprises a complementarity determining region 1 (CDR1)comprising an amino acid sequence set forth in SEQ ID NO:303;complementarity determining region 2 (CDR2) comprising an amino acidsequence set forth in SEQ ID NO: 304; and a complementarity determiningregion 3 (CDR3) comprising an amino acid sequence set forth in SEQ IDNO: 305.

222. The PD-1-binding construct of embodiment 1, wherein the at leastone VHH domain comprises a complementarity determining region 1 (CDR1)comprising an amino acid sequence set forth in SEQ ID NO:306;complementarity determining region 2 (CDR2) comprising an amino acidsequence set forth in SEQ ID NO: 307; and a complementarity determiningregion 3 (CDR3) comprising an amino acid sequence set forth in SEQ IDNO: 308.

223. The PD-1-binding construct of embodiment 1, wherein the at leastone VHH domain comprises a complementarity determining region 1 (CDR1)comprising an amino acid sequence set forth in SEQ ID NO:309;complementarity determining region 2 (CDR2) comprising an amino acidsequence set forth in SEQ ID NO: 310; and a complementarity determiningregion 3 (CDR3) comprising an amino acid sequence set forth in SEQ IDNO: 311.

224. A PD-1-binding construct, comprising at least one heavy chain onlyvariable domain (PD-1 VHH domain) comprising a complementaritydetermining region 1 (CDR1) comprising an amino acid sequence selectedset forth in SEQ ID NO: 300; a complementarity determining region 2(CDR2) comprising an amino acid sequence set forth in SEQ ID NO: 301;and a complementarity determining region 3 (CDR3) comprising an aminoacid sequence selected set forth in SEQ ID NO: 302.

225. A PD-1-binding construct, comprising at least one heavy chain onlyvariable domain (PD-1 VHH domain) comprising a complementaritydetermining region 1 (CDR1) comprising an amino acid sequence selectedset forth in SEQ ID NO: 303; a complementarity determining region 2(CDR2) comprising an amino acid sequence set forth in SEQ ID NO: 304;and a complementarity determining region 3 (CDR3) comprising an aminoacid sequence selected set forth in SEQ ID NO: 305.

226. A PD-1-binding construct, comprising at least one heavy chain onlyvariable domain (PD-1 VHH domain) comprising a complementaritydetermining region 1 (CDR1) comprising an amino acid sequence selectedset forth in SEQ ID NO: 306; a complementarity determining region 2(CDR2) comprising an amino acid sequence set forth in SEQ ID NO: 307;and a complementarity determining region 3 (CDR3) comprising an aminoacid sequence selected set forth in SEQ ID NO: 308.

227. A PD-1-binding construct, comprising at least one heavy chain onlyvariable domain (PD-1 VHH domain) comprising a complementaritydetermining region 1 (CDR1) comprising an amino acid sequence selectedset forth in SEQ ID NO: 309; a complementarity determining region 2(CDR2) comprising an amino acid sequence set forth in SEQ ID NO: 310;and a complementarity determining region 3 (CDR3) comprising an aminoacid sequence selected set forth in SEQ ID NO: 311.

228. The PD-1-binding polypeptide construct of any of embodiments220-227, wherein the PD-1 is a human PD-1.

229. The PD-1-binding polypeptide construct of any of embodiments220-228, wherein the at least one PD-1 VHH domain is humanized.

230. The PD-1-binding polypeptide construct of any of embodiments220-224 and 228-229, wherein the one or more additional binding domainsbinds to an activating receptor on an immune cell.

231. The PD-1-binding polypeptide construct of embodiment 230, whereinthe immune cell is a T cell.

232. The PD-1-binding polypeptide construct of embodiment 230 orembodiment 231, wherein the activating receptor is CD3 (CD3ε).

233. The PD-1-binding polypeptide construct of embodiment 232 that isbispecific for PD-1 and CD3.

234. The PD-1-binding polypeptide construct of embodiment 233, whereinthe immune cell is a Natural Killer (NK) cell.

235. The PD-1-binding polypeptide construct of embodiment 230 orembodiment 234, wherein the activating receptor is CD16 (CD16a).

236. The PD-1-binding polypeptide construct of embodiment 235 that isbispecific for PD-1 and CD16a.

237. The PD-1-binding polypeptide construct of any of embodiments220-224 and 228-229, wherein the one or more additional domains binds toa tumor associated antigen (TAA).

238. The PD-1-binding polypeptide construct of any of embodiments220-224 and 228-229, wherein the one or more additional binding domainbinds to a cytokine receptor.

239. The PD-1-binding polypeptide construct of any of embodiments220-224 and 228-238, wherein the one or more additional binding domaincomprises an antibody or antigen-binding fragment thereof.

240. The PD-1-binding polypeptide construct of any of embodiments220-224 and 228-239, wherein the one or more additional binding domainis monovalent.

241. The PD-1-binding polypeptide construct of embodiment 240, whereinthe antibody or antigen-binding fragment thereof is an Fv, adisulfide-stabilized Fv (dsFv), scFv, a Fab, a single domain antibody(sdAb), a VNAR, or a VHH.

242. The PD-1-binding polypeptide construct of embodiment 238, whereinthe one or more additional binding domain is a cytokine or is atruncated fragment or variant thereof capable of binding to the cytokinereceptor.

243. The PD-1-binding polypeptide construct of embodiment 242, whereinthe cytokine is an interferon that is a type I interferon or a type IIinterferon, is a truncated fragment or variant of a type I interferon oris a truncated fragment or variant of a type II interferon.

244. The PD-1-binding polypeptide construct of embodiment 243, wherein:

the type I interferon is an IFN-alpha or an IFN-beta or is a truncatedfragment or variant thereof; or

the type II interferon is an IFN-gamma or is a truncated fragment orvariant thereof.

245. The PD-1-binding polypeptide construct of any of embodiments220-244, wherein the polypeptide comprises an immunoglobulin Fc region.

246. The PD-1-binding polypeptide construct of any of embodiments220-224 and 228-245, wherein the polypeptide comprises an immunoglobulinFc region that links the at least one single domain antibody and the oneor more additional binding domain.

247. The PD-1-binding polypeptide construct of any of embodiments220-246 that is a dimer.

248. The PD-1-binding polypeptide construct of any of embodiments245-247, wherein the Fc region is a homodimeric Fc region.

249. The PD-1-binding polypeptide construct of any of embodiments245-248, wherein the Fc region comprises the sequence of amino acids setforth in any of SEQ ID NOS: 8, 10, 11, 12 or 13, or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ IDNOS: 8, 10, 11, 12 or 13.

250. The PD-1-binding polypeptide construct of any of embodiments245-248, wherein the Fc region is a human IgG1.

251. The PD-1-binding polypeptide construct of embodiment 250, whereinthe Fc region comprises the sequence of amino acids set forth in SEQ IDNO:8 or a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO: 8.

252. The PD-1-binding polypeptide construct of any of embodiments245-247, wherein the Fc region is a heterodimeric Fc region.

253. The PD-1-binding polypeptide construct of any of embodiments245-252, wherein the Fc region exhibits effector function.

254. The PD-1-binding polypeptide construct of any of embodiments245-253 wherein the Fc region comprises a polypeptide comprising one ormore amino acid modification that reduces effector function and/orreduces binding to an effector molecule selected from an Fc gammareceptor or C1q.

255. The PD-1-binding polypeptide construct of embodiment 254, whereinthe one or more amino acid modification is deletion of one or more ofGlu233, Leu234 or Leu235.

256. The PD-1-binding polypeptide construct of embodiment 254 orembodiment 255, wherein the Fc region comprises the sequence of aminoacids set forth in SEQ ID NO:9 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9.

257. The PD-1-binding polypeptide construct of any of embodiments220-256, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in any of SEQ ID NOS: 296-299, or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS:296-299 and binds PD-1.

258. The PD-1-binding polypeptide construct of any of embodiments220-257, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:296, (ii) a humanized variant of SEQ IDNO:296, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:296 and binds PD-1.

259. The PD-1-binding polypeptide construct of any of embodiments220-258, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in SEQ ID NO:296.

260. The PD-1-binding polypeptide of any of embodiments 220-258, whereinthe at least one PD-1 VHH domain comprises a CDR1 comprising an aminoacid sequence set forth in SEQ ID NO: 300; a CDR2 comprising an aminoacid sequence set forth in SEQ ID NO: 301; and a CDR3 comprising anamino acid sequence set forth in SEQ ID NO: 302.

261. The PD-1-binding polypeptide construct of any of embodiments220-258 and 260 36, wherein the at least one PD-1 VHH domain comprises aCDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 300, 301, and 302,respectively.

262. The PD-1-binding polypeptide construct of any of embodiments220-257, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:297, (ii) a humanized variant of SEQ IDNO:297, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:297 and binds PD-1.

263. The PD-1-binding polypeptide construct of any of embodiments220-257 and 262, wherein the at least one PD-1 VHH domain comprises thesequence set forth in SEQ ID NO:297.

264. The PD-1-binding polypeptide of any of embodiments 220-257 and 262,wherein the at least one PD-1 VHH domain comprises a CDR1 comprising anamino acid sequence set forth in SEQ ID NO: 303; a CDR2 comprising anamino acid sequence set forth in SEQ ID NO: 304; and a CDR3 comprisingan amino acid sequence set forth in SEQ ID NO: 305.

265. The PD-1-binding polypeptide construct of any of embodiments220-257, 262, and 264 wherein the at least one PD-1 VHH domain comprisesa CDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 303, 304, and 305,respectively.

266. The PD-1-binding polypeptide construct of any of embodiments220-257, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:298, (ii) a humanized variant of SEQ IDNO:298, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:298 and binds PD-1.

267. The PD-1-binding polypeptide construct of any of embodiments220-257 and 266, wherein the at least one PD-1 VHH domain comprises thesequence set forth in SEQ ID NO:297.

268. The PD-1-binding polypeptide of any of embodiments 220-257 and 266,wherein the at least one PD-1 VHH domain comprises a CDR1 comprising anamino acid sequence set forth in SEQ ID NO: 306; a CDR2 comprising anamino acid sequence set forth in SEQ ID NO: 307; and a CDR3 comprisingan amino acid sequence set forth in SEQ ID NO: 308.

269. The PD-1-binding polypeptide construct of any of embodiments220-257, 266 and 268, wherein the at least one PD-1 VHH domain comprisesa CDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 306, 307, and 308,respectively.

270. The PD-1-binding polypeptide construct of any of embodiments220-257, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:299, (ii) a humanized variant of SEQ IDNO:299, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:299 and binds PD-1.

271. The PD-1-binding polypeptide construct of any of embodiments220-257 and 270, wherein the at least one PD-1 VHH domain comprises thesequence set forth in SEQ ID NO:299.

272. The PD-1-binding polypeptide of any of embodiments 220-257 and 270,wherein the at least one PD-1 VHH domain comprises a CDR1 comprising anamino acid sequence set forth in SEQ ID NO: 309; a CDR2 comprising anamino acid sequence set forth in SEQ ID NO: 310; and a CDR3 comprisingan amino acid sequence set forth in SEQ ID NO: 311.

273. The PD-1-binding polypeptide construct of any of embodiments220-257, 270, and 272, wherein the at least one PD-1 VHH domaincomprises a CDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 309, 310, and311, respectively.

274. The PD-1-binding polypeptide construct of any of embodiments220-258, 260-262, 264-266, 268-270, and 272-273, wherein the at leastone PD-1 VHH domain comprises least 95% sequence identity to SEQ ID NO:296-299 and binds PD-1.

275. The PD-1-binding polypeptide construct of any of embodiments220-258, 260-262, 264-266, 268-270, and 272-274, wherein the at leastone PD-1 VHH domain is set forth in SEQ ID NO: 296-299.

276. The multispecific polypeptide construct of any of embodiments60-99, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in any of SEQ ID NOS: 296-299, or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOS:296-299 and binds PD-1.

277. The multispecific polypeptide construct of any of embodiments 60-99or 276, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:296, (ii) a humanized variant of SEQ IDNO:296, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:296 and binds PD-1.

278. The multispecific polypeptide construct of any of embodiments60-99, 276, or 277, wherein the at least one PD-1 VHH domain comprisesthe sequence set forth in SEQ ID NO:296.

279. The multispecific polypeptide construct of any of embodiments60-99, 276 or 277, wherein the at least one PD-1 VHH domain comprises aCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 300; aCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 301; anda CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 302.

280. The multispecific polypeptide construct of any of embodiments60-99, 276, 277 or 279, wherein the at least one PD-1 VHH domaincomprises a CDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 300, 301, and302, respectively.

281. The multispecific polypeptide construct of any of embodiments 60-99or 276, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:297, (ii) a humanized variant of SEQ IDNO:297, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:297 and binds PD-1.

282. The multispecific polypeptide construct of any of embodiments60-99, 276, or 281, wherein the at least one PD-1 VHH domain comprisesthe sequence set forth in SEQ ID NO:297.

283. The multispecific polypeptide construct of any of embodiments60-99, 276, or 281, wherein the at least one PD-1 VHH domain comprises aCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 303; aCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 304; anda CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 305.

284. The multispecific polypeptide construct of any of embodiments60-99, 276, 281 or 283, wherein the at least one PD-1 VHH domaincomprises a CDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 303, 304, and305, respectively.

285. The multispecific polypeptide construct of any of embodiments 60-99or 276, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:298, (ii) a humanized variant of SEQ IDNO:298, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:298 and binds PD-1.

286. The multispecific polypeptide construct of any of embodiments60-99, 276, or 285, wherein the at least one PD-1 VHH domain comprisesthe sequence set forth in SEQ ID NO:297.

287. The multispecific polypeptide construct of any of embodiments60-99, 276, or 285, wherein the at least one PD-1 VHH domain comprises aCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 306; aCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 307; anda CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 308.

288. The multispecific polypeptide construct of any of embodiments60-99, 276, 285 or 287, wherein the at least one PD-1 VHH domaincomprises a CDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 306, 307, and308, respectively.

289. The multispecific polypeptide construct of any of embodiments 60-99or 276, wherein the at least one PD-1 VHH domain comprises the sequenceset forth in (i) SEQ ID NO:299, (ii) a humanized variant of SEQ IDNO:299, or (iii) a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:299 and binds PD-1.

290. The multispecific polypeptide construct of any of embodiments60-99, 276, or 289, wherein the at least one PD-1 VHH domain comprisesthe sequence set forth in SEQ ID NO:299.

291. The multispecific polypeptide construct of any of embodiments60-99, 276, or 289, wherein the at least one PD-1 VHH domain comprises aCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 309; aCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 310; anda CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 311.

292. The multispecific polypeptide construct of any of embodiments60-99, 276, 289, or 291 wherein the at least one PD-1 VHH domaincomprises a CDR1, CDR2 and CDR3 set forth in SEQ ID NOS: 300, 301, and302, respectively.

293. The multispecific polypeptide construct of any of embodiments 60-99or 276-292, wherein the at least one PD-1 VHH domain comprises asequence of amino acids that exhibits at least 95% sequence identity toSEQ ID NO: 296, 297, 298, or 299 and binds PD-1.

294. The multispecific polypeptide construct of any of embodiments 60-99or 276-292, wherein the at least one PD-1 VHH domain is set forth in SEQID NO: 296, 297, 298, or 299.

295. The multispecific polypeptide construct of any of embodiments276-294, wherein one or both of the first and second componentscomprises at least one co-stimulatory receptor binding region (CRBR)that binds a co-stimulatory receptor.

296. The multispecific polypeptide construct of embodiment 295, whereinthe at least one co-stimulatory receptor binding region (CRBR) ispositioned amino-terminally relative to the Fc region and/orcarboxy-terminally relative to the CD3 binding region of themultispecific polypeptide construct.

297. The multispecific polypeptide construct of embodiment 295 orembodiment 296, wherein the multispecific polypeptide constructcomprises only one co-stimulatory receptor binding region (CRBR).

298. The multispecific polypeptide construct of any of embodiments295-297, wherein the multispecific polypeptide construct comprises twoco-stimulatory receptor binding region (CRBR), optionally which are thesame or different.

299. The multispecific polypeptide construct of any of embodiments295-297, wherein the at least one co-stimulatory receptor binding region(CRBR) is or comprises the extracellular domain or binding fragmentthereof of the native cognate binding partner of the co-stimulatoryreceptor, or a variant thereof that exhibits binding activity to theco-stimulatory receptor.

300. The multispecific polypeptide construct of any of embodiments295-298, wherein the at least one co-stimulatory receptor binding region(CRBR) is an antibody or antigen-binding fragment thereof selected fromthe group consisting of a Fab fragment, a F(ab′)2 fragment, an Fvfragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody,and a single domain light chain antibody.

301. The multispecific polypeptide construct of embodiment 300, whereinthe antibody or antigen-binding fragment thereof is a Fv, a scFv, a Fab,a single domain antibody (sdAb), a VNAR, or a VHH.

302. The multispecific polypeptide construct of embodiment 300 orembodiment 301, wherein the antibody or antigen-binding fragment is ansdAb.

303. The multispecific polypeptide construct of embodiment 302, whereinthe sdAb is a human or humanized sdAb.

304. The multispecific polypeptide construct of any of embodiments295-303, wherein the at least one co-stimulatory receptor binding region(CRBR) binds a co-stimulatory receptor selected from among 41BB (CD137),OX40 (CD134), CD27, glucocorticoid-induced TNFR-related protein (GITR),CD28, ICOS, CD40, B-cell activating factor receptor (BAFF-R), B-cellmaturation antigen (BCMA), Transmembrane activator and CAML interactor(TACI), and NKG2D.

305. The multispecific polypeptide construct of any of embodiments295-304, wherein the at least one co-stimulatory receptor binding region(CRBR) binds a co-stimulatory receptor selected from among 41BB (CD137),OX40 (CD134), and glucocorticoid-induced TNFR-related protein (GITR).

306. The multispecific polypeptide construct of any of embodiments295-305, wherein the at least one co-stimulatory receptor binding region(CRBR) comprises the sequence of amino acids set forth in SEQ ID NO:210or a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequenceset forth in SEQ ID NO:210 and binds 4-1BB.

307. The multispecific polypeptide construct of any of embodiments276-306, wherein the multispecific polypeptide construct comprises afirst and a second antigen binding domains that binds to a TAA.

308. The multispecific polypeptide construct of embodiment 307, whereinthe antigen binding domains bind to the same tumor-associated antigen(TAA).

309. The multispecific polypeptide construct of embodiment 307 orembodiment 308, wherein the first antigen binding domain and the secondantigen binding domain binds a different or nonoverlapping epitope ofthe same TAA and/or compete for binding to the same TAA.

310. The multispecific polypeptide construct of embodiment 307, whereinthe first antigen binding domain and the second antigen binding domainbind a different TAA.

311. The multispecific polypeptide construct of any of embodiments307-310, wherein one antigen binding domain is positionedamino-terminally relative to the Fc region and one antigen bindingdomain is positioned carboxy-terminally relative to the CD3 bindingregion.

312. The multispecific polypeptide construct of any of embodiments276-311, wherein the antigen binding domain that binds a TAA, orindependently each of the antigen binding domains that binds a TAA,comprises an extracellular domain or binding fragment thereof of thenative cognate binding partner of the TAA, or a variant thereof thatexhibits binding activity to the TAA.

313. The multispecific polypeptide construct of any of embodiments276-311, wherein the antigen binding domain that binds a TAA, orindependently each of the antigen binding domains that binds a TAA, isan antibody or antigen-binding fragment thereof.

314. The multispecific polypeptide construct of embodiment 313, whereinthe antibody or antigen-binding fragment thereof is a Fv, a scFv, a Fab,a single domain antibody (sdAb), a VNAR, or a VHH.

315. The multispecific polypeptide construct of embodiment 313 orembodiment 314, wherein the antibody or antigen-binding fragment is ansdAb.

316. The multispecific polypeptide construct of embodiment 315, whereinthe sdAb is a human or humanized sdAb.

317. The multispecific polypeptide construct of any of embodiments276-316, wherein the antigen binding domain, or independently each ofthe antigen binding domains, binds to a tumor antigen selected fromamong 1-92-LFA-3, 5T4, Alpha-4 integrin, Alpha-V integrin, alpha4beta1integrin, alpha4beta7 integrin, AGR2, Anti-Lewis-Y, Apelin J receptor,APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9,(Lewis a), Carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20,CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD40L, CD41, CD44,CD44v6, CD47, CD51, CD52, CD56, CD64, CD70, CD71, CD74, CD80, CD81,CD86, CD95, CD117, CD123, CD125, CD132, (IL-2RG), CD133, CD137, CD138,CD166, CD172A, CD248, CDH6, CEACAM5 (CEA), CEACAM6 (NCA-90), CLAUDIN-3,CLAUDIN-4, cMet, Collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10,CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL3, DLL4, DPP-4, DSG1,EDA, EDB, EGFR, EGFRviii, Endothelin B receptor (ETBR), ENPP3, EpCAM,EPHA2, EPHB2, ERBB3, F protein of RSV, FAP, FGF-2, FGF8, FGFR1, FGFR2,FGFR3, FGFR4, FLT-3, Folate receptor alpha (FRα), GAL3ST1, G-CSF,G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GP IIb/IIIa receptors,Gp130, GPIIB/IIIA, GPNMB, GRP78, HER2/neu, HER3, HER4, HGF, hGH, HVEM,Hyaluronidase, ICOS, IFNalpha, IFNbeta, IFNgamma, IgE, IgE Receptor(FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL-12R,IL-12Rbeta1, IL13, IL13R, IL15, IL17, IL18, IL21, IL23, IL23R,IL27/IL27R (wsx1), IL29, IL-31R, IL31/IL31R, IL2R, IL4, IL4R, IL6, IL6R,Insulin Receptor, Jagged Ligands, Jagged 1, Jagged 2, KISS1-R, LAG-3,LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, Mesothelin,MRP4, MUC1, Mucin-16 (MUC16, CA-125), Na/K ATPase, NGF, Nicastrin, NotchReceptors, Notch 1, Notch 2, Notch 3, Notch 4, NOV, OSM-R, OX-40, PAR2,PDGF-AA, PDGF-BB, PDGFRalpha, PDGFRbeta, PD-1, PD-L1, PD-L2,Phosphatidyl-serine, P1GF, PSCA, PSMA, PSGR, RAAG12, RAGE, SLC44A4,Sphingosine 1 Phosphate, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFbeta,TIGIT, TIM-3, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TMEM31, TNFalpha,TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, Transferrin, Transferrin receptor,TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, and WISP-3.

318. The multispecific polypeptide construct of any of embodiments310-317, wherein:

the first polypeptide comprises in order of N-terminus to C-terminus afirst antigen binding domain that binds a TAA, a first polypeptidecomprising the first Fc polypeptide of the heterodimeric Fc region, thelinker, the VH or VL domain of the anti-CD3 antibody or antigen bindingfragment and a second antigen binding domain that binds a TAA; and

the second polypeptide comprises in order of N-terminus to C-terminusone of the PD-1 VHH domain or the CRBR, the second Fc polypeptide of theheterodimeric Fc region, the linker, optionally the same linker aspresent in the first polypeptide, the other of the VH or VL domain ofthe anti-CD3 antibody or antigen binding fragment, and the other of thePD-1 VHH domain or the CRBR.

319. The multispecific polypeptide construct of any of embodiments276-318, wherein the linker is a peptide or polypeptide linker,optionally wherein the linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acids in length.

320. The multispecific polypeptide construct of any of embodiments276-319, wherein the linker is a non-cleavable linker.

321. The multispecific polypeptide construct of embodiment 320, whereinthe non-cleavable linker comprises GS, GGS, GGGGS (SEQ ID NO:125),GGGGGS (SEQ ID NO:126) and combinations thereof.

322. The multispecific polypeptide construct of any of embodiments276-321, wherein the linker is or comprises the sequenceGGGGGSGGGGGSGGGGGS (SEQ ID NO:127).

323. The multispecific polypeptide construct of any of embodiments276-319, wherein the linker is a cleavable linker.

324. The multispecific polypeptide construct of embodiment 323, whereinthe cleavable linker is a polypeptide that functions as a substrate fora protease.

325. The multispecific polypeptide construct of embodiment 324, whereinthe protease is produced by an immune effector cell, by a tumor, or bycells present in the tumor microenvironment.

326. The multispecific polypeptide construct of embodiment 324 orembodiment 325, wherein the protease is produced by an immune effectorcell and the immune effector cell is an activated T cell, a naturalkiller (NK) cell, or an NK T cell.

327. The multispecific polypeptide construct of any of embodiments324-326, wherein the protease is selected from among matriptase, amatrix metalloprotease (MMP), granzyme B, and combinations thereof.

328. The multispecific polypeptide construct of embodiment 327, whereinthe protease is granzyme B.

329. The multispecific polypeptide construct of any of embodiments324-328, wherein the cleavable linker comprises the amino acid sequenceGGSGGGGIEPDIGGSGGS (SEQ ID NO:171).

330. An isolated single domain antibody that binds PD-1, comprising acomplementarity determining region 1 (CDR1) comprising an amino acidsequence set forth in SEQ ID NO: 300; a complementarity determiningregion 2 (CDR2) comprising an amino acid sequence set forth in SEQ IDNO: 301; and a complementarity determining region 3 (CDR3) comprising anamino acid sequence set forth in SEQ ID NO: 302.

331. The isolated single domain antibody of embodiment 330, comprisingthe amino acid sequence set forth in SEQ ID NO:296, or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any ofSEQ ID NOS: 296 and binds PD-1.

332. The isolated single domain antibody of embodiment 330 or embodiment331, wherein the single domain antibody comprises the sequence set forthin (i) SEQ ID NO:296, (ii) a humanized variant of SEQ ID NO:296, or(iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:296 and binds PD-1.

333. The isolated single domain antibody of any of embodiments 330-332,wherein the sdAb comprises the sequence set forth in SEQ ID NO:296.

334. The isolated single domain antibody of any of embodiments 330-332,wherein the sdAb comprises a CDR1 comprising an amino acid sequence setforth in SEQ ID NO: 300; a CDR2 comprising an amino acid sequence setforth in SEQ ID NO: 301; and a CDR3 comprising an amino acid sequenceset forth in SEQ ID NO: 302.

335. The isolated single domain antibody of any of embodiments 330-332and 334, wherein the sdAb comprises a CDR1, CDR2 and CDR3 set forth inSEQ ID NOS: 300, 301, and 302, respectively.

336. An isolated single domain antibody that binds PD-1, comprising acomplementarity determining region 1 (CDR1) comprising an amino acidsequence set forth in SEQ ID NO: 303; a complementarity determiningregion 2 (CDR2) comprising an amino acid sequence set forth in SEQ IDNO: 304; and a complementarity determining region 3 (CDR3) comprising anamino acid sequence set forth in SEQ ID NO: 305.

337. The isolated single domain antibody of embodiment 336, comprisingthe amino acid sequence set forth in SEQ ID NO:297, or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any ofSEQ ID NOS: 297 and binds PD-1.

338. The isolated single domain antibody of embodiment 336 or embodiment337, wherein the single domain antibody comprises the sequence set forthin (i) SEQ ID NO:297, (ii) a humanized variant of SEQ ID NO:297, or(iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:297 and binds PD-1.

339. The isolated single domain antibody of any of embodiments 336-338,wherein the sdAb comprises the sequence set forth in SEQ ID NO:297.

340. The isolated single domain antibody of any of embodiments 336-338,wherein the sdAb comprises a CDR1 comprising an amino acid sequence setforth in SEQ ID NO: 303; a CDR2 comprising an amino acid sequence setforth in SEQ ID NO: 304; and a CDR3 comprising an amino acid sequenceset forth in SEQ ID NO: 305.

341. The isolated single domain antibody of any of embodiments 336-338and 340, wherein the sdAb comprises a CDR1, CDR2 and CDR3 set forth inSEQ ID NOS: 303, 304, and 305, respectively.

342. An isolated single domain antibody that binds PD-1, comprising acomplementarity determining region 1 (CDR1) comprising an amino acidsequence set forth in SEQ ID NO: 306; a complementarity determiningregion 2 (CDR2) comprising an amino acid sequence set forth in SEQ IDNO: 307; and a complementarity determining region 3 (CDR3) comprising anamino acid sequence set forth in SEQ ID NO: 308.

343. The isolated single domain antibody of embodiment 342, comprisingthe amino acid sequence set forth in SEQ ID NO:298, or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any ofSEQ ID NOS: 298 and binds PD-1.

344. The isolated single domain antibody of embodiment 342 or embodiment343, wherein the single domain antibody comprises the sequence set forthin (i) SEQ ID NO:298, (ii) a humanized variant of SEQ ID NO:298, or(iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:298 and binds PD-1.

345. The isolated single domain antibody of any of embodiments 342-344,wherein the sdAb comprises the sequence set forth in SEQ ID NO:298.

346. The isolated single domain antibody of any of embodiments 342-344,wherein the sdAb comprises a CDR1 comprising an amino acid sequence setforth in SEQ ID NO: 306; a CDR2 comprising an amino acid sequence setforth in SEQ ID NO: 307; and a CDR3 comprising an amino acid sequenceset forth in SEQ ID NO: 308.

347. The isolated single domain antibody of any of embodiments 342-344and 346, wherein the sdAb comprises a CDR1, CDR2 and CDR3 set forth inSEQ ID NOS: 306, 307, and 308, respectively.

348. An isolated single domain antibody that binds PD-1, comprising acomplementarity determining region 1 (CDR1) comprising an amino acidsequence set forth in SEQ ID NO: 309; a complementarity determiningregion 2 (CDR2) comprising an amino acid sequence set forth in SEQ IDNO: 310; and a complementarity determining region 3 (CDR3) comprising anamino acid sequence set forth in SEQ ID NO: 311.

349. The isolated single domain antibody of embodiment 348, comprisingthe amino acid sequence set forth in SEQ ID NO:299, or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any ofSEQ ID NOS: 299 and binds PD-1.

350. The isolated single domain antibody of embodiment 348 or embodiment349, wherein the single domain antibody comprises the sequence set forthin (i) SEQ ID NO:299, (ii) a humanized variant of SEQ ID NO:299, or(iii) a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:299 and binds PD-1.

351. The isolated single domain antibody of any of embodiments 348-350,wherein the sdAb comprises the sequence set forth in SEQ ID NO:299.

352. The isolated single domain antibody of any of embodiments 348-350,wherein the sdAb comprises a CDR1 comprising an amino acid sequence setforth in SEQ ID NO: 309; a CDR2 comprising an amino acid sequence setforth in SEQ ID NO: 310; and a CDR3 comprising an amino acid sequenceset forth in SEQ ID NO: 311.

353. The isolated single domain antibody of any of embodiments 348-350and 352, wherein the sdAb comprises a CDR1, CDR2 and CDR3 set forth inSEQ ID NOS: 309, 310, and 311, respectively.

354. The isolated single domain antibody of any of embodiments 330-332,334-338, 340-344, 346-350, and 352-353, wherein the sdAb comprises thesequence of amino acids set forth in any one of SEQ ID NOS: 296-299.

355. The isolated single domain antibody of any of embodiments 330-332,334-338, 340-344, 346-350, and 352-354, wherein the sdAb comprises thesequence of amino acids set forth in SEQ ID NO: 296, 297, 298, or 299.

356. A polynucleotide(s) encoding the PD-1-binding polypeptide of any ofembodiments 220-275.

357. A polynucleotide(s) encoding the multispecific polypeptideconstruct of any of embodiments 276-329.

358. A polynucleotide, comprising a first nucleic acid sequence encodinga first polypeptide of a multispecific construct of any of embodiments276-329 and a second nucleic acid sequence encoding a second polypeptideof the multispecific construct, wherein the first and second nucleicacid sequence are separated by an internal ribosome entry site (IRES),or a nucleic acid encoding a self-cleaving peptide or a peptide thatcauses ribosome skipping.

359. The polynucleotide of embodiment 358, wherein the first nucleicacid sequence and second nucleic acid sequence are operably linked tothe same promoter.

360. The polynucleotide of embodiment 359, wherein the nucleic acidencoding a self-cleaving peptide or a peptide that causes ribosomeskipping is selected from a T2A, a P2A, a E2A or a F2A.

361. A polynucleotide encoding the single domain antibody of any ofembodiments 330-355.

362. A vector, comprising the polynucleotide of any of embodiments356-361.

363. The vector of embodiment 362 that is an expression vector.

364. The vector of embodiment 362 or embodiment 363 that is a viralvector or a eukaryotic vector, optionally wherein the eukaryotic vectoris a mammalian vector.

365. A cell, comprising polynucleotide or polynucleotides of any ofembodiments 356-361, or a vector or vectors of any of embodiments362-364.

366. The cell of embodiment 365, wherein the cell is recombinant orisolated.

367. The cell of embodiment 366, wherein the cell is a mammalian cell.

368. A method of producing a polypeptide, the method comprisingintroducing into a cell a polynucleotide or polynucleotides of any ofembodiments 356-361 or a vector or vectors of any of embodiments 362-364and culturing the cell under conditions to produce the multispecificpolypeptide construct.

369. The method of embodiment 368, further comprising isolating orpurifying the polypeptide from the cell.

370. A polypeptide produced by the method of embodiment 368 orembodiment 369.

371. An engineered immune cell, comprising a binding molecule comprisingthe binding molecule of any of embodiments 220-275 or the single domainantibody of any of embodiments 330-355, optionally wherein the bindingmolecule is secretable from the cell.

372. The engineered cell of embodiment 371, further comprising achimeric antigen receptor (CAR).

373. The engineered immune cell of embodiment 371 or embodiment 372,wherein the cell is a lymphocyte.

374. The engineered immune cell of any of embodiments 372-373, whereinthe cell is a T cell or a natural killer (NK) cell.

375. The engineered immune cell of any of embodiments 372-374, whereinthe CAR comprises an extracellular domain comprising an antigen bindingdomain that binds a TAA, a transmembrane domain; and an intracellularsignaling domain.

376. The engineered immune cell of embodiment 375, wherein theintracellular signaling domain comprises an immunoreceptortyrosine-based activation motif (ITAM) signaling domain, optionallywherein the intracellular signaling domain is or comprises a CD3zetasignaling domain, optionally a human CD3zeta signaling domain.

377. The engineered immune cell of embodiment 376, wherein theintracellular signaling domain further comprises a signaling domain of acostimulatory molecule.

378. A pharmaceutical composition comprising the PD-1-bindingpolypeptide of any of embodiments 220-275, the multispecific polypeptideconstruct of any of embodiments 276-329, the single domain antibody ofany of embodiments 330-355 or the engineered immune cell of any ofembodiments 371-377.

379. The pharmaceutical composition of embodiment 378, comprising apharmaceutically acceptable carrier.

380. The pharmaceutical composition of embodiment 378 or embodiment 379that is sterile.

381. A method of stimulating or inducing an immune response in asubject, the method comprising administering, to a subject in needthereof, the PD-1-binding polypeptide of any of embodiments 220-275, themultispecific polypeptide construct of any of embodiments 276-329, thesingle domain antibody of any of embodiments 330-355 or the engineeredimmune cell of any of embodiments 371-377 or a pharmaceuticalcomposition of embodiment 378-380.

382. The method of embodiment 381, wherein the immune response isincreased against a tumor or cancer, optionally a tumor or a cancer thatexpresses PD-1.

383. The method of embodiment 381 or embodiment 382, wherein the methodtreats a disease or condition in the subject.

384. A method of treating a disease or condition in a subject, themethod comprising administering, to a subject in need thereof, atherapeutically effective amount of the PD-1-binding polypeptide of anyof embodiments 220-275, the multispecific polypeptide construct of anyof embodiments 276-329, the single domain antibody of any of embodiments330-355 or the engineered immune cell of any of embodiments 371-377 or apharmaceutical composition of embodiment 378-380.

385. The method of embodiment 384 or embodiment 385, wherein the diseaseor condition is a tumor or a cancer.

386. The method of any of embodiments 381-385, wherein said subject is ahuman.

VIII. Examples

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Generation of PD-1 sdAb

Single domain antibodies targeting human PD-1 were generated viaimmunization of llamas and alpaca. Llamas and alpaca were immunized witha recombinant version of the human PD-1 extracellular domain (ECD; aminoacids 25-167 of human PD-1 set forth in SEQ ID NO:286, e.g. UniProt No.Q15116) set forth as follows:

LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLNPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRSAGQFQ

Following the development of specific anti-PD1 antibody titers,llama/alpaca peripheral blood mononuclear cells (PBMCs) were isolatedfrom 500 mL of blood from the immunized animal and total mRNA wasisolated using the Qiagen RNeasy Maxi Kit and subsequently converted tofirst strand cDNA using Thermo Superscript IV Reverse Transcriptase andoligo-dT priming. Single domain antibody (sdAb; also called VHH)sequences were specifically amplified via PCR using the cDNA as templateand cloned into a yeast surface display vector as sdAb-Fc-AGA2 fusionproteins. The Fc was a human IgG1 Fc (set forth in SEQ ID NO:8).

Yeast libraries displaying these sdAbs were enriched using recombinantforms of the PD-1 ECD via magnetic bead isolation followed byfluorescence activated cell sorting (FACS). Sorted yeast were plated outand isolated colonies were picked into 96-well blocks and grown in mediathat switched the expression from surface displayed sdAb-Fc to secretioninto the media. Exemplary identified sdAbs are set forth in Table E1.

TABLE E1 PD-1 sdAbs Clone VHH name CDR1 SEQ ID NO CDR2 SEQ ID NO CDR3SEQ ID NO SEQ ID NO 18H10 GSVTGANTMG 272 LIGNYVTH 278 YTDNLGTS 283 2841-14 GRTVSIYAMG 313 GIGWNGGTTY 314 ESAAGTLGDY 315 312

Example 2: Humanization of Camelid Derived PD-1 sdAb

Exemplary camelid derived PD-1 sdAb, 18H10, were humanized using thehuman VH3-23 germline as scaffold. Camelid residues that contribute tosolubility, specificity, stability and/or affinity remained unmodified.In addition all humanized variants contained the modification ofLeu11Glu (L11E) and the carboxy-terminal modifications of Ser112Lys(S112K) and Ser113Pro (S113P) as these are known prevent or reduce therecognition of pre-existing ADA directed toward sdAbs (as described inUS20160207981).

Table E2 sets forth exemplary PD-1 sdAbs humanized variants.

TABLE E2 PD-1 sdAbs Humanized Variants VHH Clone name CDR1 SEQ ID NOCDR2 SEQ ID NO CDR3 SEQ ID NO SEQ ID NO 18H10 Humanized Variantshz18H10v1 GSMTGANTMG 268 LIGNYVTH 278 YTDNLGTS 283 251 hz18H10v2GSMTGANTMG 268 LIGNYVTH 278 YTDNLGTS 283 252 hz18H10v3 GSMTGANTMG 268LIGNYVTH 278 YTDNLGTS 283 253 hz18H10v4 GSMTGANTMG 268 LIGNYVTH 278YTDNLGTS 283 254 hz18H10v5 GSMTGANTMG 268 LIGNYVTH 278 YTDNLGTS 283 255hz18H10v6 GSVTGANTMG 272 LIGNYVTH 278 YTDNLGTS 283 256 hz18H10v7GSITGANTMG 273 LIGNYVTH 278 YTDNLGTS 283 257 hz18H10v8 GSVTGANTMG 272LIGNYVTH 278 YTDNLGTS 283 258 hz18H10v9 GSVTGANTMG 272 LIGNYVTH 278YTDNLGTS 283 259 hz18H10v10 GSMTGANTMG 268 LIGNYVTH 278 YTDNLGTS 283 260hz18H10v11 GSVTGANTMG 272 LIGNYVTH 278 YTDNLGTS 283 261 hz18H10v12GSVTGANTMG 272 LIGNYVTH 278 YTDNLGTS 283 262 hz18H10v13 GSMTGANTMG 268LIGNYVTH 278 YTDNLGTS 283 263 hz18H10v14 GSVTGANTMG 272 LIGNYVTH 278YTDNLGTS 283 264 hz18H10v15 GSITGANTMG 273 LIGNYVTH 278 YTDNLGTS 283 265hz18H10v16 GSITGANTMG 273 LIGNYVTH 278 YTDNLGTS 283 266 hz18H10v17GSITGANTMG 273 LIGNYVTH 278 YTDNLGTS 283 267

Example 3: Binding of sdAb to PD-1 Expressing Cells by Flow Cytometry

Specificity and relative affinity were assessed for purified sdAb-Fcs onPD-1-expressing cells. For transient transfection of 293 cells,freeStyle 293 cells were resuspended at 1×10⁶ cells per mL in freshFreeStyle 293 expression medium. Cells were seeded into 50 mL pertransfection and incubated on a shaker at 37° C. while transfectionreagents were prepared. 50 μg of each transfection plasmid were dilutedinto 500 μL of OptiMEM. In a separate tube for each transfection, 150 μgof polyethylenimine (PEI; 75 μL of a 2 mg/mL solution) were added to 500μL of OptiMEM and then mixed 1:1 with the DNA:OptiMEM solution. DNA andPEI were complexed for 15 minutes at room temperature. DNA:PEI complexeswere then added drop-wise to a prepared flask of FreeStyle 293 cells andmixed by swirling. Transfected cells were incubated overnight in a 37°C. shaker to allow time for protein expression. Transfection plasmidsused encoded citrine-tagged full-length PD1 proteins of human,cynomolgus and murine origin.

Binding of exemplary PD-1-sdAb-Fc fusion proteins described in Examples1 and 2 were assessed by flow cytometry using the transientlytransfected PD-1-expressing cells. Untransfected FreeStyle 293 cells ortransiently transfected FreeStyle 293 cells were diluted to 0.5×10×10⁶cells/mL in FACS Buffer (lx TBS, 0.01% FBS, 0.002% Sodium Azide) andplated at 100 μL/well in a 96-well round bottom assay plate. The assayplates were centrifuged at about 750 rpm for 5 minutes, then thesupernatants were removed and primary antibody dilutions were added asfollows. A titration diluted antibody was added to the assay platescontaining 293 cells. Cells were incubated in the antibody dilutions for30 minutes at 4° C. After the 30-minute incubation, the assay plateswere centrifuged at about 750 rpm for 5 minutes, washed with 150 μL FACSBuffer, and centrifuged again at about 750 rpm for 5 minutes. The washwas removed and 50 μL/well of Alexa Fluor 647 conjugateddonkey-Anti-Human IgG diluted 1:1000 in FACS buffer was added andincubated for 20 minutes at 4° C. Assay plates were then centrifuged at750 rpm for 5 minutes, washed with 150 μL FACS Buffer, and centrifugedagain at 750 rpm for 5 minutes. The wash was removed and the cells wereresuspended in 30 μL/well of FACS Buffer for analysis by flow cytometry(iQue Intellicyte).

Exemplary results are set forth in FIG. 1A-FIG. 1G for 18H10 (parentalderived from llama; SEQ ID NO:284), humanized variants of 18H10 (SEQ IDNOs: 251-257, 259-260, and 262-266) or 1-14 (SEQ ID NO: 312) for bindinghuman PD-1 expressing cells (huPD1-FL 293), cynomolgus PD-1 expressingcells (cynoPD1-FL 293), mouse PD-1 expressing cells (muPD1-FL 293) ornon-expressing (UT 293) cells.

Example 4: Assessment of Binding of PD-1 sdAb to Activated Human T Cellsby Flow Cytometry

Binding of PD-1-sdAb-Fc fusion proteins to activated human T cells wasassessed by flow cytometry.

For enrichment and activation of human T cells, Peripheral bloodmononuclear cells (PBMCs) were isolated from human donor blood usingdensity gradient centrifugation. Blood samples were diluted with PBS/2%FBS (1:2) and 30 mL of diluted blood was layered onto 15 mL ofLymphoprep density gradient medium. After centrifugation, the PBMC layerat the interphase of plasma and Lymphoprep was removed and remaining redblood cells were lysed using red blood cell lysis buffer for 5 minutesat room temperature. Non-T cell populations were labeled withbiotinylated anti-lineage marker antibodies against CD14, CD16, CD19,CD20, CD36, CD56, CD123, TCR γ/δ (20 minutes, room temperature) anddepleted using magnetic streptavidin particles. The unbound cellsupernatant containing the T cell fraction was retained. Enriched humanT cells were activated for 3 days by plating them at a density of about2×10⁶ cells per mL media in tissue culture plates coated with 1 μg/mLmouse anti-human CD3 (OKT3). Activated T cells were washed once in PBSbefore further use in binding assays.

Binding was assessed and quantified by flow cytometry substantially asdescribed in Example 3, except for binding to activated human T cells, afour-fold, 10-point serial dilution of 18H10 (SEQ ID NO: 284), humanized18H10 (hzv7; SEQ ID NO: 257), or 1-14 (SEQ ID NO: 312) ranging from 167nM to 0.00847 nM was used for incubation, As shown in FIG. 2, the testedexemplary PD-1-targeting construct and the humanized variant were foundto bind the enriched and activated human T cells.

Example 5: Assessment of PD-1/PD-L1 Blockade Using a Reporter Assay

A PD-1-expressing Jurkat effector reporter cell line, in which TCRengagement leads to the transcription of a luciferase reporter gene, wasused to assess the ability of exemplary sdAbs targeting PD-1 to blockthe interaction of PD-1 and PD-L1. In the assay, PD-L1-expressingaAPC/CHOK1 cells were co-cultured with the Jurkat reporter cells toprovide a TCR-specific activation signal, while simultaneouslysuppressing this signal through the engagement of PD-1 on the effectorcell. The ability of PD-1 sdAb to block the suppressed signal, andenhance TCR engagement, was monitored.

PD-L1 expressing aAPC/CHOK1 cells were plated in 100 μl Ham's F12supplemented with 10% FBS one day before the assay. On the day of theassay, all media was discarded and replaced with 40 μL of assay media(RPMI 1640, supplemented with 1% FBS) containing titrations of the testproteins containing 18H10 (SEQ ID NO: 41) or humanized 18H10 (hzv7; SEQID NO: 14) (starting concentration: 50 nM, titrated 1:4). Jurkat PD-1reporter cells were then added to the plates (40 μL) and the plate wasincubated for 6 h (37° C., 5% CO2 in a humidified atmosphere). After theincubation, an equal volume of BioGlo Luciferase Assay Substrate wasadded to the wells and incubated for 10 minutes at room temperature andluminescence was assessed and analyzed.

As shown in FIG. 3A and FIG. B, blockade of PD-1/PD-L1 by the exemplarytested proteins 18H10 (SEQ ID NO: 284), humanized 18H10 (hzv7; SEQ IDNO: 257) or 1-14 (SEQ ID NO: 312) was observed as indicated by thepresence of TCR engagement and luciferase transcription.

Example 6: Method of Producing TAA-Targeted Constrained CD3 BindingProteins with Anti-PD1 sdAbs

Multispecific polypeptide constructs were generated containing adisulfide stabilized anti-CD3 Fv binding region that exhibitsconstrained CD3 binding, a heterodimeric Fc domain, one or more TAAantigen binding domains positioned amino-terminally relative to the Fcregion and/or carboxy-terminally relative to the CD3 binding region, andan inhibitory receptor binding region (IRBR) containing a single domainantibody (sdAb) against PD-1, which was positioned amino-terminallyrelative to the Fc region and/or carboxy-terminally relative to the CD3binding region. In some cases, the multispecific polypeptide constructswere generated to contain at least one co-stimulatory receptor bindingregion (CRBR), e.g. against 41BB, which was positioned amino-terminallyrelative to the Fc region and/or carboxy-terminally relative to the CD3binding region.

In the exemplary constructs, polynucleotides encoding at least a firstpolypeptide chain and a second polypeptide chain of the heterodimericmultispecific polypeptide construct were generated and cloned into aplasmid for expression. The first polypeptide chain generally includedin order, from the N-terminus to C-terminus, an Fc hole polypeptide(e.g. set forth in SEQ ID NO:112, or in some cases SEQ ID NO:114); acleavable or a non-cleavable linker, such as one containing one or moresubstrate recognition sites for a protease; and a variable light (VL)domain of a dsFv anti-CD3 antibody (e.g. set forth in SEQ ID NO:285).The second polypeptide chain generally included in order, from theN-terminus to C-terminus, an Fc knob polypeptide (e.g. set forth in SEQID NO: 105, or in some cases SEQ ID NO:109); the same cleavable linkeror the same non-cleavable linker as the first polypeptide chain; and avariable heavy domain of a dsFv anti-CD3 antibody (e.g. set forth in SEQID NO:47). The constructs were generated with the exemplarynon-cleavable linker, GGGGGSGGGGGSGGGGGS (SEQ ID NO:127), or theexemplary cleavable linker, GGSGGGGIEPDIGGSGGS (SEQ ID NO:171)containing a substrate recognition site for granzyme B. One or both ofthe polypeptide chains additionally encoded PD-1 sdAb (e.g. SEQ IDNO:284 or SEQ ID NO: 257) amino terminal to the Fc domain and/or carboxyterminal to the CD3 binding region, and/or a 4-1BB sdAb (e.g. SEQ IDNO:210) co-stimulatory receptor binding domain amino terminal to the Fcdomain and/or carboxy terminal to the CD3 binding region, in variousconfigurations.

Separate plasmids encoding each chain of a heterodimeric constrained CD3binding protein were transiently transfected at an equimolar ratio intomammalian cells (either HEK293 or CHO) using polyethylenimine.Recombinant protein secreted into the supernatant was collected after3-7 days, and secreted recombinant protein was purified by protein Achromatography, followed by either preparative size exclusionchromatography (SEC) or flow-through hydrophobic interactionchromatography (HIC). Heterodimeric protein was selectively purifiedowing to a mutation designed into one chain of the heterodimeric Fc atposition I253R or H435R (usually the hole-Fc) such that it did not bindprotein A. The second chromatography step on SEC (AKTA with Superdex-200resin) or FT-HIC (AKTA with butyl/phenyl sepharose) was used to removeundesired cross-paired species containing two heterodimeric Fcs thatwere more hydrophobic and twice the expected molecular weight.

The method favored production of heterodimeric multispecific polypeptideconstructs, containing properly paired species of heterodimeric Fc andthe disulfide stabilized anti-CD3 Fv as described (e.g. anti-CD3 VH withthe mutation G44C as set forth in SEQ ID NO: 47 and VL with the mutationG100C as set forth in SEQ ID NO: 285). Purified heterodimericconstrained CD3 binding protein was stable and did not accumulatecross-paired species upon prolonged incubation at 4° C. or increasedprotein concentration.

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

Sequences # SEQUENCE ANNOTATION   1 GGSGGS (GGS)2 linker   2 GGSGGSGGS(GGS)3 linker   3 GGSGGSGGSGGS (GGS)4 linker   4 GGSGGSGGSGGSGGS(GGS)5 linker   5 GGGG glycine linker   6 GGGGG glycine linker   7GGGGGG glycine linker   8PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV human IgG1 FcDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALPAPIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMHEALHNHYTQK SLSLSPGK   9PAPGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV Fc xELLEVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWESNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK 10 PAPPVAGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD human IgG2 FcGVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPAPIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVEWESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHEALHNHYTQKS LSLSPGK  11PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFKWYV human IgG3 FcDGVEVHNAKT KPREEQYNST FRVVSVLTVL HQDWLNGKEY KCKVSNKALPAPIEKTISKT KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAVEWESSGQPEN NYNTTPPMLD SDGSFFLYSK LTVDKSRWQQ GNIFSCSVMHEALHNRFTQK SLSLSPGK  12PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV human IgG4 FcDGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLPSSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMHEALHNHYTQK SLSLSLGK  13PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV human IgG4 FcDGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLPSSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAVEWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMHEALHNHYTQK SLSLSLGK  14 EPKSSDKTHTCPPC modified IgG1 hinge  15 DKTHTCPPCtruncated IgG1 hinge  16 ESKYGPPCPPC modified IgG4 hinge  17GQGTLVTVKPGG carboxy-terminal sequence  18 GQGTLVTVEPGG carboxy-terminalsequence  19 QVQLVQSGGG VVQPGRSLRL SCKASGYTFT RYTMHWVRQA PGKGLEWIGYOKT3 VH INPSRGYTNY NQKVKDRFTI SRDNSKNTAF LQMDSLRPED TGVYFCARYYDDHYCLDYWG QGTPVTVSS  20DIQMTQSPSS LSASVGDRVT ITCSASSSVS YMNWYQQTPG KAPKRWIYDT OKT3 VLSKLASGVPSR FSGSGSGTDY TFTISSLQPE DIATYYCQQW SSNPFTFGQG TKLQIT  21QVQLVQSGGG VVQPGRSLRL SCKASGYTFT RYTMHWVRQA PGKGLEWIGY OKT3 humanized VHINPSRGYTNY NQKVKDRFTI SRDNSKNTAF LQMDSLRPED TGVYFCARYYDDHYSLDYWG QGTPVTVSS  22DVQLVQSGAE VKKPGASVKV SCKASGYTFT RYTMHWVRQA PGQGLEWIGY OKT3 humanized VHINPSRGYTNY ADSVKGRFTI TTDKSTSTAY MELSSLRSED TATYYCARYYDDHYCLDYWG QGTTVTVSS  23QVQLVQSGAE LKKPGASVKV SCKASGYTFT RYTMHWVRQA PGQCLEWMGY OKT3 humanized VHINPSRGYTNY NQKFKDKATL TADKSTSTAY MELRSLRSDD TAVYYCARYYDDHYSLDYWG QGTLVTVSS  24QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT OKT3 humanized VLSKLASGVPAH FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEIN  25DIQMTQSPSS LSASVGDRVT ITCRASQSVS YMNWYQQKPG KAPKRWIYDT OKT3 humanized VLSKVASGVPAR FSGSGSGTDY SLTINSLEAE DAATYYCQQW SSNPLTFGGG TKVEIK  26DIQLTQSPSI LSASVGDRVT ITCRASSSVS YMNWYQQKPG KAPKRWIYDT OKT3 humanized VLSKVASGVPYR FSGSGSGTEY TLTISSMQPE DFATYYCQQW SSNPLTFGCG TKVEIKRT  27EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 HvATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSA  28QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAP anti-CD3 LvGVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGGGTKLTVL  29 TYAMNanti-CD3 VH CDR1  30 RIRSKYNNYATYYADSVKD anti-CD3 VH CDR2  31HGNFGNSYVSWFAY anti-CD3 VH CDR3  32 RSSTGAVTTSNYAN anti-CD3 VL CDR1  33GTNKRAP anti-CD3 VL CDR2  34 ALWYSNLWV anti-CD3 VL CDR3  35EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNY anti-CD3 VH1ATYYADSVKDRFTISRDDSKNSLYLQMNSLKIEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  36EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH2ATYYADSVKDRFTISRDDSKSSLYLQMNNLKIEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  37EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH3ATYYADSVKDRFTISRDDSKSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  38EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH4ATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  39EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VHSATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  40EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNY anti-CD3 VH6ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  41EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVGRIRSKYNNY anti-CD3 VH7ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWG QGTLVTVSS  42EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH8ATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG QGTLVTVS  43EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH9ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTTVTVSS  44EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH10ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWG QGTTVTVSS  45EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH11ATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  46EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH12ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVKP  47EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH13ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVKP  48EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH14ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG CGTLVTVKP  49EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNY anti-CD3 VH15ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  50EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVSRIRSKYNNY anti-CD3 VH16ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  51EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNY anti-CD3 VH17ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  52EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH18ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  53EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVSRIRSKYNNY anti-CD3 VH19ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  54EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVSRIRSKYNNY anti-CD3 VH20ATYYADSVKGRFTISRDDAKNTLYLQMSSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  55EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVGRIRSKYNNY anti-CD3 VH21ATYYADSVKDRFTISRDDSKNSLYLQMNSLKIEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  56EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH22ATYYADSVKDRFTISRDDSKSSLYLQMNNLKIEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  57EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH23ATYYADSVKDRFTISRDDSKSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  58EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH24ATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  59EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH25ATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  60EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKCLEWVSRIRSKYNNY anti-CD3 VH26ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  61EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKCLEWVGRIRSKYNNY anti-CD3 VH27ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWG QGTLVTVSS  62EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH28ATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG QGTLVTVS  63EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH29ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWG QGTTVTVSS  64EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH30ATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWG QGTTVTVSS  65EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNY anti-CD3 VH31ATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS  66QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAP anti-CD3 VL1GVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGGGTKLTVL  67QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIGGTNKRAP anti-CD3 VL2GVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGCGTKLEIK  68QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP anti-CD3 VL3WTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVL  69QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAP anti-CD3 VL4GVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL  70QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAP anti-CD3 VL5GVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL  71QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAP anti-CD3 VL6GVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL  72QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAP anti-CD3 VL7GTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL  73QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAP anti-CD3 VL8GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL  74QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGSGSLLGGKAA anti-CD3 VL9LTISGAQPEDEADYYCALWYSNHWVFGGGTKLEIK  75QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIGGTNKRAP anti-CD3 VL10GVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGCGTKLEIK  76QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQCFRGLIGGTNKRAP anti-CD3 VL11GVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGEGTKLEIK  77QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAP anti-CD3 VL12GVPARFSGSLIGDKAALTITGAQIEDEAIYFCALWYSNLWVFGCGTKLTVL  78QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIGGTNKRAP anti-CD3 VL13GVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGGGTKLEIK  79QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP anti-CD3 VL14WTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGCGTKLTVL  80QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAP anti-CD3 VL15GVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL  81QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAP anti-CD3 VL16GVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVFGCGTKLTVL  82QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAP anti-CD3 VL17GVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGCGTKLTVL  83QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAP anti-CD3 VL18GTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGCGTKLTVL  84QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAP anti-CD3 VL19GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGCGTKLTVL  85QVQLQESGGG LVQAGGSLRL SCAASGRTFS NYHMGWFRQA PGKERELVAA anti-CD3 VHHISGSGGSTYY TDSVKGRFTI SRNNAKNTMS LQMSNLKPED TGVYYCTTPTEKGSSIDYWG QGTQVTVSSG RYPYDVPDY  86QLQLQESGGGLVQPGGSLRLSCAASGFTLDNYAIGWFRQAPGKEREGVSCISSSDGST FR alpha sdAbYYADSVKGRFTISRNNAKGTVYLLMNSLKPEDTAVYYCATELVPACTYSNGRGPLDGM DYWGKGTQVTVKP 87 EVQLLESGGGEVQPGGSLRLSCAASGSIFSIDATAWYRQAPGKQRELVAIITSSGSTNFR alpha sdAb YPESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCNAITRYGGSTYDFWGQGTLVTVKP  88 EVQPGGSLRLSCAASETFGVVFTLGWYRQAPGKGREFVARVTGTDTVDYAESVKGRFTFR alpha sdAb ISSDFARNTVYLQMNSLRAEDTAVYYCNTGAYWGQGTLVTVKP  89EVQLVESGGGLVQPGGSLRLSCAASGFILDYYAIGWFRQAPGKEREGVLCIDASDDIT cMET sdAbYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCATPIGLSSSCLLEYDYDYWG QGTLVTVKP  90QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDT CD20 Sc FVDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSGSGGGGSGGGGTGGGGSDIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK  91QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWINWVRQA PGQGLEWMGR CD20 VHIFPGDGDTDY NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNVFDGYWLVYWG QGTLVTVSS  92 DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYWCD20 VL YLQKPGQSPQLLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGVYYCAQNLELPYTFGGGTKVE IKRTV  93QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVYYSGTTN DLL3 scFvYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTF GGGTKLEIK  94QVQLQESGPG LVKPSETLSL TCTVSGGSIS SYYWSWIRQP DLL3 scFvPGKGLEWIGYVYYSGTTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAADTAVYYCASIAV TGFYFDYWGQ GTLVTVSSGG GGSGGGGSGG GGSEIVLTQSPGTLSLSPGERVTLSCRASQ RVNNNYLAWY QQRPGQAPRL LIYGASSRATGIPDRFSGSG SGTDFTLTIS RLEPEDFAVY YCQQYDRSPL TFGGGTKLEI K  95QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMAWINTYTGEP DLL3 FdTYADDFKGRFAFSLETSASTASLQIINLKNEDTATYFCARIGDSSPSDYWGQGTTLTVSSSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC  96SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVVWYQQKPGQSPKLLIYYASNRYTGV DLL3 LCPDRFAGSGYGTDFSFTISTVQAEDLAVYFCQQDYTSPWTFGGTKLEIRRTVAAPSGVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  97EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 5T4 FdFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC  98DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWASTRLTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS 5T4 LCLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC  99EVQLVESGGGL VQPKGSLKLS CAASGFTFNT YAMNWVRQAP GKGLEWVARIRSKSNNYATY YADSVKDRFT ISRDDSQSML YLQMNNLKTE DTAMYXCVRQ anti-5T4 VHWDYDVRAMNY WGQGTSVTVS S 100DIVMTQSHIF MSTSVGDRVS ITCKASQDVDTAVAWYQQKP GQSPKLLIYWASTRLTGVPD RFTGSGSGTD FTLTISNVQSEDLADYFCQQ anti-5T4 VL YSSYPYTFGGGTKLEIK101 QVQLQESGPGLVKPSQTLSLTCTVSGGSISSFNYYWSWIRHHPGKGLEWIGYIYYSGSTYSNPSLKSRVTISVDTSKNQFSLTLSSVTAADTAVYYCARGYNWNYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA gpNMB FdVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 102EIVMTQSPATLSVSPGERATLSCRASQSVDNNLVWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY gpNMB LCSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 103DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY Knob FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPT 104DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKFNWY Hole FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPT 105DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG Knob FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPT 106DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKFNWYVDG Hole FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPT 107DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY Knob FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 108DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKFNWY Hole FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 109DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG Knob FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 110DKTHTCPPCPAPGGPSVFLFPPKPKDTLMRSRTPEVTCVVVDVSHEDPEVKFNWYVDG Hole FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 111DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY Hole FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPT 112DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG Hole FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPT 113DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY Hole FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG 114DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG Hole FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPG 115DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWY Knob FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPT 116DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDG Knob FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPT 117DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWY Knob FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPG 118DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDG Knob FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVVHEALHNHYTQKSLSLSPG 119DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWY Hole FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPT 120DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDG Hole FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPT 121DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWY Hole FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPG 122DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVDVSHEDPEVKFNWYVDG Hole FcVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVVHEALHNRYTQKSLSLSPG 123(GGGGS)n, wherein n is 1 to 5 Linker 124 (GGGGGS)n, wherein n is 1 to 4linker 125 GGGGS Linker 126 GGGGGS Linker 127 GGGGGSGGGGGSGGGGGS Linker128 GGGGSGGGGSGGGGS Linker 129 GGSGGGGSGGGGSGGGGS Linker 130GlyxXaa-Glyy-Xaa-Glyz LinkerXaa is independently selected from A, V, L, I, M,F, W, P,G, S, T, C, Y, N,Q, K, R, H, D, or Ex, y, and z are each integers in the range from 1-5 131Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly LinkerXaa is independently selected from A, V, L, I, M,F, W, P,G, S, T, C, Y, N,Q, K, R, H, D, or E 132 (SSSSG)n Linker n = 1-9 133GGGGG-C-GGGGG Linker 134 (EAAAK)n Linker 135 AS-(AP)n-GT Linker n = 2-20136 AS-(EAAAK)n-GT Linker n = 2-20 137 (GGGGA)n Linker n = 2-20 138(PGGGS)n Linker n = 2-20 139 (AGGGS)n Linker n = 2-20 140GGS-(EGKSSGSGSESKST)n-GGS Linker n = 2-20 141 SSSASASSA Linker 142GSPGSPG Linker 143 ATTTGSSPGPT Linker 144X1 X2 X3 X4 X5 (P4 P3 P2 P1 ↓ P1′) Linker consensusX1 = I, L, Y, M, F, V, or A; (P4 = I, L, Y, M, F, V, or A)X2 = A, G, S, V, E, D, Q, N, or Y; (P3 = A, G, S, V, E, D, Q, N, or Y)X3 = H, P, A, V, G, S, or T; (P2 = H, P, A, V, G, S, or T)X4 = D or E; (P1 = D or E)X5 = I, L, Y, M, F, V, T, S, G or A (P1′ = I, L, Y, M, F,V, T, S, G or A) 145 X1 E X3 D X5 (P4 P3 P2 P1 ↓ P1′) Linker consensusX1 = I or L; (P4 = I or L) (P3 = E) X3 = P or A; (P2 = P or A)X5 = I, V, T, S, or G (P1′ = I, V, T, S, or G) 146 LEADgranzyme B substrate 147 LEPD Linker 148 LEAE Linker 149 IEPDI Linker150 LEPDG Linker 151 LEADT Linker 152 IEPDG Linker 153 IEPDV Linker 154IEPDS Linker 155 IEPDT Linker 156 X1QARX5 (P1QAR↓(A/V)) Linker consensusX1 = any amino acid; (P1 is any amino acid) X5 = A or V 157RQARX5 (RQAR(A/V)) Linker X5 = A or V 158 RQAR matriptase substrate 159RQARV linker 160 X1X2 X3 X4 (P3 P2 P1 ↓ P1′) Linker consensusX1 = P, V or A; (P3 = P, V or A) X2 = Q or D; (P2 = Q or D)X3 = A or N; (P1 = A or N) X4 = L, I or M (P1′ = L, I or M) 161PX2X3X4 (P3 P2 P1 ↓ P1′) Linker consensus (P3 = P)X2 = Q or D; (P2 = Q or D) X3 = A or N; (P1 is A or N)X4 = L or I (P1′ is L on) 162 PAGL MMP substrate 163 TGLEADGSPAGLGRQARVGLinker 164 TGLEADGSRQARVGPAGLG Linker 165 TGSPAGLEADGSRQARVGS Linker 166TGPAGLGLEADGSRQARVG Linker 167 TGRQARVGLEADGSPAGLG Linker 168TGSRQARVGPAGLEADGS Linker 169 TGPAGLGSRQARVGLEADGS Linker 170GPAGLGLEPDGSRQARVG Linker 171 GGSGGGGIEPDIGGSGGS Linker 172GGSGGGGLEADTGGSGGS Linker 173 GSIEPDIGS Linker 174 GSLEADTGS Linker 175GGSGGGGIEPDGGGSGGS Linker 176 GGSGGGGIEPDVGGSGGS Linker 177GGSGGGGIEPDSGGSGGS Linker 178 GGSGGGGIEPDTGGSGGS Linker 179 GGGSLEPDGSGSLinker 180 GPAGLGLEADGSRQARVG Linker 181 GGEGGGGSGGSGGGS Linker 182GSSAGSEAGGSGQAGVGS Linker 183 GGSGGGGLEAEGSGGGGS Linker 184GGSGGGGIEPDPGGSGGS Linker 185 TGGSGGGGIEPDIGGSGGS Linker 186ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL 41BBLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEANSAFGFQGRLLHLSAGQRLGVHLHIEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 187EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYT 41BB VHNYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSS 188SYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIP 41BB VLERFSGSNSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVL 189QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVT 41BB VHYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTL VTVSS 190EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI 41BB VLPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK 191QMQLVQSGAEVKKPGASVKVSCKASGYSFSGYYMHWVRQAPGQGLEWMGWVNPMSGGT 41BB VHNYAQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREGMAMRLELDKWGQGTLV TVSS 192SYELTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIP 41BB VLERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSVVFGGGTQLTVL 193QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGQAGNIKLREDKDPNKMMATIYEL41BB AnticalinKEDKSYNVTGVTFDDKKCTYAISTFVPGSQPGEFTLGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQ CIDG 194QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGQAGNIRLREDKDPIKMMATIYEL41BB AnticalinKEDKSYDVTMVKFDDKKCMYDIWTFVPGSQPGEFTLGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQ CIDG 195QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGQAGNIRLREDKDPNKNIMATIYE41BB AnticalinLKEDKSYDVTAVAFDDKKCTYDIWTFVPGSQPGEFTLGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPID QCIDG 196QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGQAGNIKLREDKDPNKNIMATIYE41BB AnticalinLKEDKSYDVTAVAFDDKKCTYDIWTFVPGSQPGEFTLGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPID QCIDG 197QDSTSDLIPA PPLSKVPLQQ NFQDNQFHGK WYVVGQAGNI KLREDSKNIMA 41BB AnticalinTIYELKEDKS YDVTGVSFDD KKCTYAIMTF VPGSQPGEFT LGKIKSFPGHTSSLVRVVST NYNQHAMVFF KFVFQNREEF YITLYGRTKE LTSELKENFIRFSKSLGLPE NHIVFPVPID QCIDG 198QDSTSDLIPA PPLSKVPLQQ NFQDNQFHGK WYVVGQAGNI KLREDKDPVK 41BB AnticalinMMATIYELKE DKSYDVTGVT FDDKKCRYDI STFVPGSQPG EFTFGKIKSFPGHTSSLVRV VSTNYNQHAM VFFKFVFQNR EEFYITLYGR TKELTSELKENFIRFSKSLG LPENHIVFPV PIDQCIDG 199QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGQAGNIRLREDKDPHKNIMATIYE41BB AnticalinLKEDKSYDVTGVTFDDKKCTYAISTFVPGSQPGEFTLGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPID QCIDG 200QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGQAGNIKLREDKDPNKNIMATIYE41BB AnticalinLKEDKSYDVTGVTFDDKKCTYAISTLVPGSQPGEFTFGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPID QCIDG 201QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGQAGNIRLREDKDPSKNIMATIYE41BB AnticalinLKEDKSYDVTAVTFDDKKCNYAISTFVPGSQPGEFTLGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPID QCIDG 202REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE71-254 of humanDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP 41BBLPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI PAGLPSPRSE203 LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYY85-254 of humanVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQ 41BBLGRLLHLSAGQRLGVHLHIEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 204DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAK80-254 of humanAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNS 41BBLAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRS E 205PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSW52-254 of humanYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHL 4-1BBLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 206REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE71-248 of humanDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP 41BBLPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI PAGL 207LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYY85-248 of humanVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQ 41BBLGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 208DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAK80-248 of humanAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNS 41BBLAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 209PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSW52-248 of humanYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHL 41BBLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 210EVQLLESGGGEVQPGGSLRLSCAASGFSFSINAMGWYRQAPGKRREFVAAIESGRNTV 41BB sdAbYAESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCGLLKGNRVVSPSVAYWGQGTL VTVKP 211QVSHRYPRIQSIKVQFIEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGY OX40 ligandFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 212QVSHRYPRFQ SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF OX40 ligandYLISLKGYFS QEVNISLHYQ KDEEPLFQLK KVRSVNSLMV ASLTYKDKVYLNVTTDNTSL DDFHVNGGEL ILIHQNPGEFCVL 213QVSHRYPRIQSIKVQFIEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGY OX40 ligandFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 214QVSHRYPRIQ SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF OX40 ligandYLISLKGYFS QEVNISLHYQ KDEEPLFQLK KVRSVNSLMV ASLTYKDKVYLNVTTDNTSL DDFHVNGGEL ILIHQNPGEF CVL 215VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYF OX40 ligandSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 216EVQLVQSGAEVKKPGASVKVSCKASGYTFTDSYMSWVRQAPGQGLEWIGDMYPDNGDS OX40 VHSYNQKFRERVTITRDTSTSTAYLELSSLRSEDTAVYYCVLAPRWYFSVWGQGTLVTVS S 217DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLRSGV OX40 VLPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPPTFGQGTKVEIKRT 218EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQASGKGLEWVGRIRSKANSY OX40 VHATAYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTSGIYDSSGYDYWGQGTL VTVSS 219DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSN OX40 VLRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK 220EVQLLESGGGEVQPGGSLRLSCAASGFTFSDAFMYWVRQAPGKGLEWVSSISNRGLKT OX40 sdAbAYAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCSRDVDGDFRGQGTLVTVKP 221QLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANY GITR ligandNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYW GIILLANPQFIS222 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISSGGTTY GITR VHYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGGYYDSMDYWGQGTLVTV SS 223EIVLTQSPGTLSLSPGERATLSCRASESVDNYGVSFMNWYQQKPGQAPRLLIYAASNQ GITR VLGSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTKEVTWTFGQGTKVEIK 224QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDD GITR VHKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVT VSS 225EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRLLIYSASYRYSGI GITR VLPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNTDPLTFGGGTKVEIK 226QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYFWSWIRQPPGKGLEWIGYIYYSGT GITR VHTYYNPSLKSRVTISIDTSKNQFSLKLSSVTAADTAVYYCARDLFYYDTSGPRGFDPWG QGTLVTVSS 227EIVLTQSPGTLSLSPGERATLSCRASQTVSSNYLAWYQQKPGQAPRLLIYGSSTRATG GITR VLIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSSPWTFGQGTKVEIK 228QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYPGSNK GITR VHYYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGELGRYYYYGMDVWGQG TTVTVSS 229DIQMTQSPSSLSASVGDRVTVTCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGV GITR VLPSRFSGSGSG1EFTLTISSLQPEDFATYYCLQHNNYPWTFGQGTKVDIK 230EVQLLESGGGEVQPGGSLRLSCAASGSVFSIDAMGWYRQAPGKQRELVAVLSGISSAK GITR sdAbYAASAPGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYADVSTGWGRDAHGYWGQGTL VTV 231UniProt no. P32970 CD70-ECD 232QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNK CD70 VHYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVT VSS 233DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGV CD70 VLPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK 234QVQLQQSGGGLVQPGGSLRLSCAASGSIFSINGMGWYRQAPGKERELVAGLTSGGSVT ICOS sdAbNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRAEIFTRTGENYYGMDYWGK GTQVTVKP 235EVQLVESGGGEVQPGGSLRLSCAASGRMFSNYAMGWFRQAPGKEREFVAAINYRRDAA CD28 sdAbDYAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCGFTYAGWASSRRDDYNYWGQ GTLVTVKP 236RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLCD3zeta signaling YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRdomain 237 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB -derivedcostimulatory domain 238 SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSCD28-derived costimulatory domain 239RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28-derivedcostimulatory domain 2 240 FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSCD2 8-derived costimulatory domain 3 241KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDIYIWAPLAGTCCD8-derived hinge GVLLLSLVITLYC and transmembmne domain 242AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCD8-derived hinge CGVLLLSLVIT and transmembmne domain 243KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW CD8 hinge andAPLAGTCGVLLLSLVIT transmembrane domain 244 GGSGGS (GGS)2 linker 245GGSGGSGGS (GGS)3 linker 246 GGSGGSGGSGGS (GGS)4 linker 247GGSGGSGGSGGSGGS (GGS)5 linker 248 GGGG glycine linker 249 GGGGGglycine linker 250 GGSGGS (GGS)2 linker 251EVQLVESGGGEVQPGGSLRLSCAASGSMTGANTMGWYRQAPGKGRDLVSLIGNYVTHY hz18H10v1AESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 252EVQLVESGGGEVQPGGSLRLSCAASGSMTGANTMGWYRQAPGKQRDLVSLIGNYVTHY hz18H10v2AESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 253EVQLVESGGGEVQPGGSLRLSCAASGSMTGANTMGWYRQAPGKQRDLVSLIGNYVTHY hz18H10v3AESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 254EVQLVESGGGEVQPGGSLRLSCAASGSMTGANTMGWYRQAPGKQRDLVALIGNYVTHY hz18H10v4AESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 255EVQLVESGGGEVQPGGSLRLSCAASGSMTGANTMGWYRQAPGKQRDLVALIGNYVTHY hz18H10v5AESVKGRFTISRENAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 256EVQLVESGGGEVQPGGSLRLSCAASGSVTGANTMGWYRQAPGKQRDLVALIGNYVTHY hz18H10v6AESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 257EVQLVESGGGEVQPGGSLRLSCAASGSITGANTMGWYRQAPGKQRDLVALIGNYVTHY hz18H10v7AESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 258EVQLVESGGGEVQPGGSLRLSCAASGSVTGANTMGWYRQAPGKQRDLVSLIGNYVTHY hz18H10v8AESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 259EVQLVESGGGEVQPGGSLRLSCAASGSVTGANTMGWYRQAPGKQRDLVSLIGNYVTHY hz18H10v9AESVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 260EVQLVESGGGEVQPGGSLRLSCAASGSMTGANTNIGWYRQAPGKQRDLVSLIGNYVTH hz18H10v10YAESVKGRFTISRENAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 261EVQLVESGGGEVQPGGSLRLSCAASGSVTGANTNIGWYRQAPGKQRDLVSLIGNYVTH hz18H10v11YAESVKGRFTISRENAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 262EVQLVESGGGEVQPGGSLRLSCAASGSVTGANTNIGWYRQAPGKQRDLVALIGNYVTH hz18H10v12YAESVKGRFTISRENAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 263EVQLVESGGGEVQPGGSLRLSCAASGSMTGANTNIGWYRQAPGKQRELVALIGNYVTH hz18H10v13YAESVKGRFTISRENAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 264EVQLVESGGGEVQPGGSLRLSCAASGSVTGANTNIGWYRQAPGKQRDLVALIGNYVTH hz18H10v14YAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 265EVQLVESGGGEVQPGGSLRLSCAASGSITGANTNIGWYRQAPGKQRDLVALIGNYVTH hz18H10v15YAESVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 266EVQLVESGGGEVQPGGSLRLSCAASGSITGANTNIGWYRQAPGKQRDLVALIGNYVTH hz18H10v16YAESVKGRFTISRENAKNTVYLQMSSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 267EVQLVESGGGEVQPGGSLRLSCAASGSITGANTNIGWYRQAPGKQRDLVALIGNYVTH hz18H10v17YAESVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 268GSMTGANTNIG CDR1 269 GGSGGSGGS (GGS)3 linker 270 GGSGGSGGSGGS(GGS)4 linker 271 GGSGGSGGSGGSGGS (GGS)5 linker 272 GSVTGANTNIG CDR1 273GSITGANTNIG CDR1 274 GGSGGS (GGS)2 linker 275 GGSGGSGGS (GGS)3 linker276 GGSGGSGGSGGS (GGS)4 linker 277 GGSGGSGGSGGSGGS (GGS)5 linker 278LIGNYVTH CDR2 279 GGSGGS (GGS)2 linker 280 GGSGGSGGS (GGS)3 linker 281GGSGGSGGSGGS (GGS)4 linker 282 GGSGGSGGSGGSGGS (GGS)5 linker 283YTDNLGTS CDR3 284QVQLVQSGGGLVQPGGSLRLSCVASGSMTGANTMGWYRQAPGKQRDLVALIGNYHYAD 18H10SVKGRFTISRENAKNTVILQMNSLNPEDTAVYYCYLYTDNLGTSWGQGTLVTVKP 285QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAFRGLIGGTNKRAPanti-CD3 VL (CON) GVPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGCGTKLTVL286 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSN PD-1TSESFVLNWYRNISPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL 296QVQLQQSGGGLVQAGGSLRVSCAASGRTFSSYGMGWFRQAPGKEREFVAAISWSGGTQ cPD1-8YYADSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCADYTDYVVYRPQEIGYWGQG TQVTVKP 297QLQLQQSGGGLAQAGGSLRLSCAASGRTVSIYAMGWFRQAPGKEREFVAGIGWNGGTT cPD1-14YYADSVEGRFTISRDHAKNTAYLQMNSLKPEDTAVYYCAAQESAAGTLGDYWGRGTQV TVKP 298QVTLKESGGGLVQAGGSLRLSCAASGRTEIIYAMGWFRQAPGKEREFVAGIGWSGGTT cPD1-13YYADSVEGRFTISRDSAKNTVYLQMRSLKPEDTAVYYCAAQDSVAGTLGDYWGQGTQV TVRP 299QVTLRESGGGLVQPGGSLRLSCAASGLTFGLYAMTWFRQAPGKDREGISCISSSDGST cPD1-5IYADSVKGRFTASRDNAKDTMYLQMNNLNPEDTAVYYCATDYETRCDYGLRLRDRTAY WGPGTQVTVKP300 GRTFSSY cPD1-8-CDR1 301 GRTVSIY cPD1-14-CDR1 302 GRTEIIYcPD1-13-CDR2 303 GLTFGLY cPD1-5-CDR1 304 SWSGGT cPD1-8-CDR2 305 GWNGGTcPD1-14-CDR2 306 GWSGGT cPD1-13-CDR2 307 SDGST cPD1-5-CDR2 308GTDYVVYRPQEIGY cPD1-8-CDR3 309 QESAAGTLGDY cPD1-14-CDR3 310 QDSVAGTLGDYcPD1-13-CDR3 311 DYETRCDYGLRLRDRTAY cPD1-5-CDR3 312QVQLQQSGGGLAQAGGSLRLSCAASGRTVSIYAMGWFRQAPGKEREFVAGIGWNGGTT 1-14YYADSVEGRFTISRDHAKNTAYLQMNSLKPEDTAVYYCAAQESAAGTLGDYWGRGTQV TVKPGG 316EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSI Anti-CD3 VHGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDSRGYGDYRLGGAYWGQG 312557TLVTVSS 317 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKCLEWVSGISWNSGSIAnti-CD3 VH GYADSVKGFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGT312557 G44C LVTVSS 318EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGI Anti-CD3 VLPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK 312557 319EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGI Anti-CD3 VLPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGCGTKVEIK 312557 Q100C 320EVQLVESGGGLVQPGRSLRLSCVASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSK CD3-VH-GDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKYGSGYGKFYHYGLDVWGQ GTTVTVSS 321EVQLVESGGGLVQPGRSLRLSCVASGFTFDDYSMHWVRQAPGKCLEWVSGISWNSGSK CD3-VH-G G44CDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKYGSGYGKFYHYGLDVWGQ GTTVTVSS 322DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV V_(K1)-39Jκ5PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK 323DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVV_(K1)-39Jκ5 Q100C PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGCGTRLEIK

1. A method of stimulating or inducing an immune response in a subject,the method comprising administering, to a subject in need thereof, aPD-1-binding polypeptide construct, comprising at least one heavy chainonly variable domain (PD-1 VHH domain) that specifically binds PD-1. 2.The method of claim 1, wherein the PD-1 binding polypeptideconstruct-comprises one or more additional binding domain that binds toa target other than PD-1.
 3. A method of treating a disease or conditionin a subject, the method comprising administering, to a subject in needthereof, a therapeutically effective amount of a PD-1-bindingpolypeptide construct, comprising at least one heavy chain only variabledomain (PD-1 VHH domain) that specifically binds PD-1.
 4. The method ofclaim 3, wherein the PD-1 binding polypeptide construct-comprises one ormore additional binding domain that binds to a target other than PD-1.5. The method of claim 1, wherein the at least one VHH domain comprisesa complementarity determining region 1 (CDR1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 268, 272, 273and 313; a complementarity determining region 2 (CDR2) comprising anamino acid sequence set forth in SEQ ID NO: 278 or 314; and acomplementarity determining region 3 (CDR3) comprising an amino acidsequence set forth in SEQ ID NO: 283 or
 315. 6. The method of claim 1,wherein the PD-1 VHH domain that specifically binds PD-1, blocks theinteraction of PD-1 and PD-L1.
 7. The method of claim 2, wherein the oneor more additional binding domain binds a co-stimulatory molecule. 8.The method of claim 2, wherein the one or more additional binding domainbinds a T-cell surface marker.
 9. The method of claim 8, wherein theT-cell surface marker is CD8.
 10. The method of claim 8, wherein theT-cell surface marker is CD4.
 11. The method of claim 2, wherein the oneor more additional binding domain binds an immune checkpoint other thanPD1.
 12. The method of claim 2, wherein the one or more additionalbinding domains binds to an activating receptor on an immune cell. 13.The method of claim 2, wherein the one or more additional domains bindsto a tumor associated antigen (TAA).
 14. The method of claim 2, whereinthe one or more additional binding domain binds to a cytokine receptor.15. The method of claim 1, wherein the polypeptide comprises animmunoglobulin Fc region.
 16. The method of claim 1, wherein the atleast one PD-1 VHH domain comprises the sequence set forth in any of SEQID NOS: 251-267, 284 or 312, or a sequence of amino acids that exhibitsat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to any of SEQ ID NOS: 251-267, 284 or312 and binds PD-1.
 17. The method of claim 1, wherein the at least onePD-1 VHH domain comprises a CDR1, CDR2 and CDR3 set forth in SEQ ID NOS:268, 278, and 283, respectively; SEQ ID NOS: 272, 278, and 283,respectively; or SEQ ID NOS: 273, 278, and 283, respectively.
 18. Themethod of claim 1, wherein the at least one PD-1 VHH domain is anisolated single domain antibody